Anti-pd-1 antibodies and uses thereof

ABSTRACT

This disclosure relates to anti-PD-1 (Programmed Cell Death Protein 1) antibodies, antigen-binding fragments, and the uses thereof.

TECHNICAL FIELD

This disclosure relates to anti-PD-1 (Programmed Cell Death Protein 1)antibodies and uses thereof.

BACKGROUND

Recent clinical and commercial success of anticancer antibodies hascreated great interest in antibody-based therapeutics in human. Clinicaltrials for human cancer that target PD-1 have achieved excellent resultsand drawn attention as a next-generation cancer therapy.

The number of cancer cases of domestic animals has also been rapidlyincreased in these days due to the increased longevity of pets andimproved diagnosis in veterinary clinic. Cancer therapies for domesticanimals, as well as those for humans, have progressed and surgical,radiation, and chemical therapies have been increasingly performed onthese domestic animals. However, the development of antibody-basedtherapeutics for veterinary use is slow. There is a need for thenext-generation cancer therapy in veterinary care.

SUMMARY

This disclosure relates to anti-PD-1 antibodies, antigen-bindingfragment thereof, and the uses thereof.

In one aspect, the disclosure provides an antibody or antigen-bindingfragment thereof that binds to PD-1 (Programmed Cell Death Protein 1)comprising:

a heavy chain variable region (VH) comprising complementaritydetermining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 regioncomprises an amino acid sequence that is at least 80% identical to aselected VH CDR1 amino acid sequence, the VH CDR2 region comprises anamino acid sequence that is at least 80% identical to a selected VH CDR2amino acid sequence, and the VH CDR3 region comprises an amino acidsequence that is at least 80% identical to a selected VH CDR3 amino acidsequence; and

a light chain variable region (VL) comprising CDRs 1, 2, and 3, whereinthe VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 regioncomprises an amino acid sequence that is at least 80% identical to aselected VL CDR2 amino acid sequence, and the VL CDR3 region comprisesan amino acid sequence that is at least 80% identical to a selected VLCDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and theselected VL CDRs, 1, 2, and 3 amino acid sequences are one of thefollowing:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12,respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18,respectively.

In some embodiments, the CDRs are in Kabat numbering. In someembodiments, the CDRs are in Chothia numbering.

In some embodiments, the antibody or antigen-binding fragmentspecifically binds to canine PD-1. In some embodiments, the antibody orantigen-binding fragment binds to PD-1 of Ailuropoda melanoleuca (giantpanda) or Felis catus (domestic cat).

In some embodiments, the antibody or antigen-binding fragment is acaninized antibody or antigen-binding fragment thereof. In someembodiments, the antibody or antigen-binding fragment is a felinizedantibody or antigen-binding fragment thereof.

In one aspect, the disclosure provides a nucleic acid comprising apolynucleotide encoding a polypeptide comprising:

-   -   (1) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising        complementarity determining regions (CDRs) 1, 2, and 3        comprising the amino acid sequences set forth in SEQ ID NOs: 1,        2, and 3, respectively, and wherein the VH, when paired with a        light chain variable region (VL) comprising the amino acid        sequence set forth in SEQ ID NO: 52, 53, 54, or 60, binds to        PD-1;    -   (2) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 4, 5, and 6,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 49,        50, 51, or 59, binds to PD-1;    -   (3) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising CDRs 1,        2, and 3 comprising the amino acid sequences set forth in SEQ ID        NOs: 7, 8, and 9, respectively, and wherein the VH, when paired        with a light chain variable region (VL) comprising the amino        acid sequence set forth in SEQ ID NO: 56, binds to PD-1;    -   (4) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 10, 11, and 12,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 55,        binds to PD-1;    -   (5) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising CDRs 1,        2, and 3 comprising the amino acid sequences set forth in SEQ ID        NOs: 13, 14, 15, respectively, and wherein the VH, when paired        with a light chain variable region (VL) comprising the amino        acid sequence set forth in SEQ ID NO: 45, 46, 47, 48 or 58 binds        to PD-1;    -   (6) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 16, 17, 18,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 42,        43, 44 or 57 binds to PD-1.

In some embodiments, the CDRs are in Kabat numbering. In someembodiments, the CDRs are in Chothia numbering.

In some embodiments, the VH when paired with a VL specifically binds tocanine PD-1, or the VL when paired with a VH specifically binds tocanine PD-1.

In some embodiments, the immunoglobulin heavy chain or the fragmentthereof is a caninized immunoglobulin heavy chain or a fragment thereof,and the immunoglobulin light chain or the fragment thereof is acaninized immunoglobulin light chain or a fragment thereof. In someembodiments, the immunoglobulin heavy chain or the fragment thereof is afelinized immunoglobulin heavy chain or a fragment thereof, and theimmunoglobulin light chain or the fragment thereof is a felinizedimmunoglobulin light chain or a fragment thereof.

In some embodiments, the antibody or antigen-binding fragment binds toPD-1 of Ailuropoda melanoleuca (giant panda) or Felis catus (domesticcat).

In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure also provides a vector comprising one ormore of the nucleic acids as described herein, and/or a vectorcomprising two of the nucleic acids as described herein. In someembodiments, the vector encodes the VL region and the VH region thattogether bind to PD-1.

In another aspect, the disclosure provides a pair of vectors, whereineach vector comprises one of the nucleic acids as described herein,wherein together the pair of vectors encodes the VL region and the VHregion that together bind to PD-1.

The disclosure also provides a cell comprising the vector as describedherein, or the pair of vectors as described herein. In some embodiments,the cell is a CHO cell.

In some embodiments, the cell has one or more of the nucleic acids asdescribed herein, or two of the nucleic acids as described herein. Insome embodiments, the two nucleic acids together encode the VL regionand the VH region that together bind to PD-1.

In one aspect, the disclosure also provides a method of producing anantibody or an antigen-binding fragment thereof. The method involve

-   -   (a) culturing the cell as described herein under conditions        sufficient for the cell to produce the antibody or the        antigen-binding fragment; and    -   (b) collecting the antibody or the antigen-binding fragment        produced by the cell.

In one aspect, the disclosure provides an antibody or antigen-bindingfragment thereof that binds to PD-1 comprising a heavy chain variableregion (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region(VL) comprising an amino acid sequence that is at least 90% identical toa selected VL sequence, wherein the selected VH sequence and theselected VL sequence are one of the following:

-   -   (1) the selected VH sequence is SEQ ID NOs: 49, 50, 51, or 59,        and the selected VL sequence is SEQ ID NOs: 52, 53, 54, or 60;    -   (2) the selected VH sequence is SEQ ID NOs: 42, 43, 44, or 57,        and the selected VL sequence is SEQ ID NOs: 45, 46, 47, 48, or        58;    -   (3) the selected VH sequence is SEQ ID NO: 55, and the selected        VL sequence is SEQ ID NO: 56.

In some embodiments, the VH comprises the sequence of SEQ ID NO: 49 andthe VL comprises the sequence of SEQ ID NO: 53. In some embodiments, theVH comprises the sequence of SEQ ID NO: 43 and the VL comprises thesequence of SEQ ID NO: 45.

In some embodiments, the antibody or antigen-binding fragmentspecifically binds to canine PD-1. In some embodiments, the antibody orantigen-binding fragment binds to PD-1 of Ailuropoda melanoleuca (giantpanda) or Felis catus (domestic cat).

In some embodiments, the antibody or antigen-binding fragment is acaninized antibody or antigen-binding fragment thereof. In someembodiments, the antibody or antigen-binding fragment is a felinizedantibody or antigen-binding fragment thereof.

In one aspect, the disclosure provides an antibody-drug conjugatecomprising the antibody or antigen-binding fragment thereof as describedherein covalently bound to a therapeutic agent. In some embodiments, thetherapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the disclosure provides a method of treating a subjecthaving cancer, the method comprising administering a therapeuticallyeffective amount of a composition comprising the antibody orantigen-binding fragment thereof as described herein, or theantibody-drug conjugate as described herein, to the subject.

In some embodiments, the subject has a solid tumor. In some embodiments,the cancer is unresectable melanoma or metastatic melanoma. In someembodiments, the cancer is non-small cell lung cancer (NSCLC), squamouscell carcinoma of the head and neck (SCCHN), head and neck cancer, renalcell carcinoma (RCC), melanoma, bladder cancer, gastric cancer,urothelial cancer, Merkel-cell carcinoma, triple-negative breast cancer(TNBC), or colorectal carcinoma.

In one aspect, the disclosure provides a method of decreasing the rateof tumor growth. The method involves contacting a tumor cell with aneffective amount of a composition comprising an antibody orantigen-binding fragment thereof as described herein, or theantibody-drug conjugate as described herein.

In one aspect, the disclosure provides a method of killing a tumor cell.The method involves contacting a tumor cell with an effective amount ofa composition comprising the antibody or antigen-binding fragmentthereof as described herein, or the antibody-drug conjugate as describedherein.

In one aspect, the disclosure provides a pharmaceutical compositioncomprising the antibody or antigen-binding fragment thereof as describedherein, and a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising the antibody drug conjugate as described herein, and apharmaceutically acceptable carrier.

In at least some aspects or embodiments, the antibody is a canine IgGantibody (e.g., a canine IgG1, IgG2, IgG3, or IgG4 antibody). In atleast some aspects or embodiments, the antibody is a feline IgGantibody.

In at least some aspects or embodiments, the antibody or antigen-bindingfragment is a single-chain variable fragment (scFV).

As used herein, the term “cancer” refers to cells having the capacityfor autonomous growth. Examples of such cells include cells having anabnormal state or condition characterized by rapidly proliferating cellgrowth. The term is meant to include cancerous growths, e.g., tumors;oncogenic processes, metastatic tissues, and malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. Also included are malignancies of the various organsystems, such as respiratory, cardiovascular, renal, reproductive,hematological, neurological, hepatic, gastrointestinal, and endocrinesystems; as well as adenocarcinomas which include malignancies such asmost colon cancers, renal-cell carcinoma, prostate cancer and/ortesticular tumors, non-small cell carcinoma of the lung, and cancer ofthe small intestine. Cancer that is “naturally arising” includes anycancer that is not experimentally induced by implantation of cancercells into a subject, and includes, for example, spontaneously arisingcancer, cancer caused by exposure of a patient to a carcinogen(s),cancer resulting from insertion of a transgenic oncogene or knockout ofa tumor suppressor gene, and cancer caused by infections, e.g., viralinfections. The term “carcinoma” is art recognized and refers tomalignancies of epithelial or endocrine tissues. The term also includescarcinosarcomas, which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures. The term “sarcoma” is art recognizedand refers to malignant tumors of mesenchymal derivation. The term“hematopoietic neoplastic disorders” includes diseases involvinghyperplastic/neoplastic cells of hematopoietic origin. A hematopoieticneoplastic disorder can arise from myeloid, lymphoid or erythroidlineages, or precursor cells thereof.

As used herein, the term “antibody” refers to any antigen-bindingmolecule that contains at least one (e.g., one, two, three, four, five,or six) complementary determining region (CDR) (e.g., any of the threeCDRs from an immunoglobulin light chain or any of the three CDRs from animmunoglobulin heavy chain) and is capable of specifically binding to anepitope. Non-limiting examples of antibodies include, e.g., monoclonalantibodies, polyclonal antibodies, multi-specific antibodies (e.g.,bi-specific antibodies), single-chain antibodies, chimeric antibodies,human antibodies, mouse antibodies, canine antibodies, felineantibodies, caninized antibodies, and felinized antibodies, etc. In someembodiments, an antibody can contain an Fc region of a canine antibody,a feline antibody, a mouse antibody, or a human antibody. The termantibody also includes derivatives, e.g., bi-specific antibodies,single-chain antibodies, diabodies, linear antibodies, andmulti-specific antibodies formed from antibody fragments.

As used herein, the term “antigen-binding fragment” refers to a portionof a full-length antibody, wherein the portion of the antibody iscapable of specifically binding to an antigen. In some embodiments, theantigen-binding fragment contains at least one variable domain (e.g., avariable domain of a heavy chain or a variable domain of light chain).Non-limiting examples of antibody fragments include, e.g., Fab, Fab′,F(ab′)₂, and Fv fragments.

The monoclonal antibodies herein specifically include “chimeric”antibodies. As used herein, the term “chimeric antibody” refers to anantibody (immunoglobulin) in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity.Typically, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, e.g., by genetic engineering, fromantibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody can be joined to canine constant segments. FIG. 2shows a schematic representation of the general structure of oneembodiment of a mouse:canine IgG. In this embodiment, the antigenbinding site is derived from mouse while the Fc portion is canine.

As used herein, the term “human antibody” refers to an antibody that isencoded by an endogenous nucleic acid (e.g., rearranged humanimmunoglobulin heavy or light chain locus) present in a human.

As used herein, the term “canine antibody” refers to an antibody that isencoded by an endogenous nucleic acid (e.g., rearranged canineimmunoglobulin heavy or light chain locus) present in a canine mammal(e.g., domestic dog). The antibody can be a naturally-occurring orrecombinantly produced immunoglobulin composed of amino acid sequencesrepresentative of natural antibodies isolated from canines of variousbreeds. Canine antibodies are antibodies having variable and constantregions derived from canine germline immunoglobulin sequences. In somecases, the canine antibodies can include amino acid residues not encodedby canine germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “canine antibody” is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto canineframework sequences.

As used herein, the term “feline antibody” refers to an antibody that isencoded by an endogenous nucleic acid (e.g., rearranged felineimmunoglobulin heavy or light chain locus) present in a feline mammal(e.g., domestic cat). The antibody can be a naturally-occurring orrecombinantly produced immunoglobulin composed of amino acid sequencesrepresentative of natural antibodies isolated from felines of variousbreeds. Feline antibodies are antibodies having variable and constantregions derived from feline germline immunoglobulin sequences. Thefeline antibodies of the disclosure can include amino acid residues notencoded by feline germline immunoglobulin sequences. However, the term“feline antibody” is not intended to include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto feline framework sequences.

As used herein, the term “caninized antibody” refers to an antibodywhich contains minimal sequence derived from a non-canine (e.g., mouse,human) immunoglobulin and contains sequences derived from a canineimmunoglobulin. In non-limiting examples, caninized antibodies arecanine immunoglobulin sequences (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-canine species (donor antibody)such as mouse having the desired specificity, affinity, and capacity. Insome instances, framework region (FR) residues of the canineimmunoglobulin sequences are replaced by corresponding non-canineresidues. Furthermore, in some embodiments, caninized antibodies caninclude residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In some embodiments, the caninized antibody includessubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-canine immunoglobulin sequence and all orsubstantially all of the FRs are those of a canine immunoglobulinsequence. The caninized antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of a canine immunoglobulin sequence. FIG. 2 showsone embodiment showing speciation or caninization of a mouse IgG. Insome embodiments, mouse CDRs are grafted onto canine frameworks.Caninized antibodies can be produced using molecular biology methodsknown in the art. Strategies for caninization of antibodies include, butare not limited to, the strategies disclosed in US20160264656; and U.S.Pat. No. 7,261,890B2; which are incorporated herein by reference in theentirety.

As used herein, the term “felinized antibody” refers to an antibodywhich contains minimal sequence derived from a non-feline (e.g., mouse,human) immunoglobulin and contains sequences derived from a felineimmunoglobulin. In non-limiting examples, felinized antibodies arefeline immunoglobulin sequences (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-feline species (donor antibody)such as mouse having the desired specificity, affinity, and capacity. Insome instances, framework region (FR) residues of the felineimmunoglobulin sequences are replaced by corresponding non-felineresidues. Furthermore, in some embodiments, felinized antibodies caninclude residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In some embodiments, the felinized antibody can includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-feline immunoglobulin sequence and all orsubstantially all of the FRs are those of a feline immunoglobulinsequence. The felinized antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of a feline immunoglobulin sequence. Felinizedantibodies can be produced using molecular biology methods known in theart.

A “parent” antibody herein is one that is encoded by an amino acidsequence used for the preparation of the variant antibody (e.g.,chimeric antibody, caninized antibody or felinized antibody). In someembodiments, the parent antibody has a canine framework region and, ifpresent, has canine antibody constant region(s). For example, the parentantibody can be a caninized or canine antibody. As another example, theparent antibody can be a felinized or feline antibody. In some otherexamples, the parent antibody is a murine monoclonal antibody or a pandaantibody.

The term “caninization” is defined as a method for transferringnon-canine antigen-binding amino acids from a donor antibody to a canineantibody acceptor framework to generate protein therapeutic treatmentsuseful in canine mammals.

The term “felinization” is defined as a method for transferringnon-feline antigen-binding amino acids from a donor antibody to a felineantibody acceptor framework to generate protein therapeutic treatmentsuseful in feline mammals.

In some embodiments, the antibody can contain minimal sequence derivedfrom an immunoglobulin of one spices (e.g., mouse, human, dog, or cat)and contains sequences derived from an immunoglobulin of Ailuropodamelanoleuca (giant panda). In non-limiting examples, the hypervariable(e.g., CDR) region residues of the recipient antibody are from anon-panda antibody (e.g., a donor antibody), e.g., a mouse, rat, orrabbit antibody, having the desired specificity, affinity, and capacity.In some embodiments, the Fv framework residues of the immunoglobulinhave amino acid resides that are derived from the immunoglobulin ofAiluropoda melanoleuca (giant panda). In some embodiments, the antibodycontains substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loops(CDRs) correspond to those of a non-panda (e.g., mouse) immunoglobulinand all or substantially all of the framework regions are those of aimmunoglobulin of giant panda. The antibody can also contain at least aportion of an immunoglobulin constant region (Fc), typically, that of animmunoglobulin of giant panda.

In some embodiments, the antibody is collected from a mammal or producedin a cell culture (e.g., hybridoma cells). In some embodiments, theantibody is produced in a non-human cell (e.g., a mouse or hamster cellline). In some embodiments, the antibody is produced in a bacterial oryeast cell. In some embodiments, the antibody is produced in atransgenic non-human animal (e.g., a bovine, a rat, or a mouse)containing an unrearranged or rearranged immunoglobulin locus (e.g.,heavy or light chain immunoglobulin locus).

As used herein, the term “single-chain antibody” refers to a singlepolypeptide that contains at least two immunoglobulin variable domains(e.g., a variable domain of a mammalian immunoglobulin heavy chain orlight chain) that is capable of specifically binding to an antigen.Non-limiting examples of single-chain antibodies are described herein.

As used herein, the term “multimeric antibody” refers to an antibodythat contains four or more (e.g., six, eight, or ten) immunoglobulinvariable domains. In some embodiments, the multimeric antibody is ableto crosslink one target molecule (e.g., PD-1) to at least one secondtarget molecule (e.g., CTLA-4) on the surface of a mammalian cell (e.g.,a canine T-cell, a feline T-cell).

As used herein, the terms “subject” and “patient” are usedinterchangeably throughout the specification and describe an animal,human or non-human, to whom treatment according to the methods of thepresent invention is provided. Veterinary and non-veterinaryapplications are contemplated by the present invention. In someembodiments, the subject is a mammal (e.g., a non-human mammal). Thesubjects can include but are not limited to mice, rats, hamsters,guinea-pigs, rabbits, ferrets, cats, dogs, giant panda, and primates.Included are, for example, non-human primates (e.g., monkey, chimpanzee,gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters,ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine,canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, when referring to an antibody, the phrases “specificallybinding” and “specifically binds” mean that the antibody interacts withits target molecule (e.g., PD-1) preferably to other molecules, becausethe interaction is dependent upon the presence of a particular structure(i.e., the antigenic determinant or epitope) on the target molecule; inother words, the reagent is recognizing and binding to molecules thatinclude a specific structure rather than to all molecules in general. Anantibody that specifically binds to the target molecule may be referredto as a target-specific antibody. For example, an antibody thatspecifically binds to a PD-1 molecule may be referred to as aPD-1-specific antibody or an anti-PD-1 antibody.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to polymers of amino acids of any lengthof at least two amino acids.

As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and“nucleic acid sequence” are used interchangeably herein to refer topolymers of nucleotides of any length of at least two nucleotides, andinclude, without limitation, DNA, RNA, DNA/RNA hybrids, andmodifications thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. Other features andadvantages of the invention will be apparent from the following detaileddescription and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a flow chart showing the first part of an exemplary protocolof making anti-dPD-1 antibodies.

FIG. 1B is a flow chart showing the second part of an exemplary protocolof making anti-dPD-1 antibodies.

FIG. 2 is a schematic representation of the general structure of amouse:canine chimeric IgG with mouse variable regions and canineconstant regions, or a caninized IgG with caninized variable regions andcanine constant regions.

FIG. 3 is a set of flow cytometry graphs showing that the anti-dPD-1antibodies block the binding between canine PD-1 (dPD-1) and caninePD-L1 (dPD-L1).

FIG. 4 is a set of graphs showing flow cytometry results of analyzingthe anti-dPD-1 antibodies' cross-reactivity with human PD-1 (hPD-1),mouse PD-1 (mPD-1), and mouse-dog chimeric PD-1 (chidPD-1). NC standsfor negative control.

FIG. 5 is a set of graphs showing flow cytometry results of analyzingthe anti-dPD-1 antibodies' cross-reactivity with panda PD-1 (pPD-1). NCstands for negative control.

FIG. 6 is a set of graphs showing flow cytometry results of analyzingthe anti-dPD-1 antibodies' cross-reactivity with feline PD-1 (cPD-1). NCstands for negative control.

FIG. 7 shows the mean of fluorescence intensity indicating the bindingactivity to panda PD-1 at different concentrations of 4A7-mHvKv-dlgG4and 1B9-mHvKv-dlgG4.

FIG. 8 shows the mean of fluorescence intensity indicating the bindingactivity to canine PD-1 at different concentrations of 4A7-mHvKv-dlgG4and 1B9-mHvKv-dlgG4.

FIG. 9 shows the mean of fluorescence intensity indicating the bindingactivity to feline PD-1 at different concentrations of 4A7-mHvKv-dlgG4and 1D8-mHvKv-dlgG4.

FIG. 10 is a graph showing the results of surface plasma resonance (SPR)using the chimeric anti-dPD-1 antibody 1D8-mHvKv-dlgG4 and canine PD-1.

FIG. 11 is a graph showing body weight over time of caninized PD-1 mice(B-dPD-1) with MC-38 tumor cells treated with chimeric and caninizedanti-dPD-1 antibodies. PS stands for physiological saline.

FIG. 12 is a graph showing percentage change of body weight over time ofcaninized PD-1 mice (B-dPD-1) with MC-38 tumor cells treated withchimeric and caninized anti-dPD-1 antibodies. PS stands forphysiological saline.

FIG. 13 is a graph showing tumor size over time in caninized PD-1 mice(B-dPD-1) with MC-38 tumor cells treated with several caninizedanti-dPD-1 antibodies. PS stands for physiological saline.

FIG. 14 is a graph showing tumor size over time in caninized PD-1 mice(B-dPD-1) with MC-38 tumor cells treated with several caninizedanti-dPD-1 antibodies. PS stands for physiological saline.

FIG. 15 is a graph showing tumor size over time in caninized PD-1 mice(B-dPD-1) with MC-38 tumor cells treated with several chimericanti-dPD-1 antibodies. PS stands for physiological saline.

FIG. 16 lists CDR sequences of mouse anti-dPD-1 antibodies (13-1B9,12-4A7, 12-1D8) and CDR sequences of related anti-dPD-1 antibodiesthereof as defined by Kabat numbering.

FIG. 17 lists CDR sequences of mouse anti-dPD-1 antibodies (13-1B9,12-4A7, 12-1D8) and CDR sequences of related anti-dPD-1 antibodiesthereof as defined by Chothia numbering.

FIG. 18 lists amino acid sequences of human PD-1 (hPD-1), canine PD-1(dPD-1), mouse PD-1 (mPD-1), monkey PD-1 (rmPD-1), chimeric PD-1(chidPD-1), panda PD-1 (pPD-1), and feline PD-1 (cPD-1).

FIG. 19 lists amino acid sequences of heavy chain variable regions andlight chain variable regions of caninized anti-dPD-1 antibodies based on1D8.

FIG. 20 lists amino acid sequences of heavy chain variable regions andlight chain variable regions of caninized anti-dPD-1 antibodies based on1B9.

FIG. 21 lists the amino acid sequence of the heavy chain variableregions and light chain variable regions of mouse anti-dPD-1 antibodies13-1B9, 12-4A7, and 12-1D8.

FIG. 22 lists the amino acid sequence of the constant regions of canineantibodies and feline antibodies.

DETAILED DESCRIPTION

The present disclosure provides examples of antibodies, antigen-bindingfragment thereof, that bind to PD-1 (Programmed Cell Death Protein 1;also known as CD279).

PD-1 and Cancer

The immune system can differentiate between normal cells in the body andthose it sees as “foreign,” which allows the immune system to attack theforeign cells while leaving the normal cells alone. This mechanismsometimes involves proteins called immune checkpoints. Immunecheckpoints are molecules in the immune system that either turn up asignal (co-stimulatory molecules) or turn down a signal.

Checkpoint inhibitors can prevent the immune system from attackingnormal tissue and thereby preventing autoimmune diseases. Many tumorcells also express checkpoint inhibitors. These tumor cells escapeimmune surveillance by co-opting certain immune-checkpoint pathways,particularly in T cells that are specific for tumor antigens (Creelan,Benjamin C. “Update on immune checkpoint inhibitors in lung cancer.”Cancer Control 21.1 (2014): 80-89). Because many immune checkpoints areinitiated by ligand-receptor interactions, they can be readily blockedby antibodies against the ligands and/or their receptors.

PD-1 (programmed death-1) is an immune checkpoint and guards againstautoimmunity through a dual mechanism of promoting apoptosis (programmedcell death) in antigen-specific T-cells in lymph nodes whilesimultaneously reducing apoptosis in regulatory T cells(anti-inflammatory, suppressive T cells).

PD-1 is mainly expressed on the surfaces of T cells and primary B cells;two ligands of PD-1 (PD-L1 and PD-L2) are widely expressed inantigen-presenting cells (APCs). The interaction of PD-1 with itsligands plays an important role in the negative regulation of the immuneresponse. Inhibition the binding between PD-1 and its ligand can makethe tumor cells exposed to the killing effect of the immune system, andthus can reach the effect of killing tumor tissues and treating cancers.

PD-L1 is expressed on the neoplastic cells of many different cancers. Bybinding to PD-1 on T-cells leading to its inhibition, PD-L1 expressionis a major mechanism by which tumor cells can evade immune attack. PD-L1over-expression may conceptually be due to 2 mechanisms, intrinsic andadaptive. Intrinsic expression of PD-L1 on cancer cells is related tocellular/genetic aberrations in these neoplastic cells. Activation ofcellular signaling including the AKT and STAT pathways results inincreased PD-L1 expression. In primary mediastinal B-cell lymphomas,gene fusion of the MHC class II transactivator (CIITA) with PD-L1 orPD-L2 occurs, resulting in overexpression of these proteins.Amplification of chromosome 9p23-24, where PD-L1 and PD-L2 are located,leads to increased expression of both proteins in classical Hodgkinlymphoma. Adaptive mechanisms are related to induction of PD-L1expression in the tumor microenvironment. PD-L1 can be induced onneoplastic cells in response to interferon 7. In microsatelliteinstability colon cancer, PD-L1 is mainly expressed on myeloid cells inthe tumors, which then suppress cytotoxic T-cell function.

The use of PD-1 blockade to enhance anti-tumor immunity originated fromobservations in chronic infection models, where preventing PD-1interactions reversed T-cell exhaustion. Similarly, blockade of PD-1prevents T-cell PD-1/tumor cell PD-L1 or T-cell PD-1/tumor cell PD-L2interaction, leading to restoration of T-cell mediated anti-tumorimmunity.

A detailed description of PD-1, and the use of anti-PD-1 antibodies totreat cancers are described, e.g., in Topalian et al. “Safety, activity,and immune correlates of anti-PD-1 antibody in cancer.” New EnglandJournal of Medicine 366.26 (2012): 2443-2454; Hirano, Fumiya, et al.“Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancertherapeutic immunity.” Cancer research 65.3 (2005): 1089-1096; Raedler,Lisa A. “Keytruda (pembrolizumab): first PD-1 inhibitor approved forpreviously treated unresectable or metastatic melanoma.” American health& drug benefits 8. Spec Feature (2015): 96; Kwok, Gerry, et al.“Pembrolizumab (Keytruda).” (2016): 2777-2789; US 20170247454; U.S. Pat.Nos. 9,834,606 B; and 8,728,474; each of which is incorporated byreference in its entirety.

The present disclosure provides several anti-PD-1 antibodies (e.g.,caninized anti-dPD-1 antibodies), antigen-binding fragments thereof, andmethods of using these anti-PD-1 antibodies and antigen-bindingfragments to inhibit tumor growth and to treat cancers in variousmammals, including canine animals (e.g., dogs), feline animals (e.g.,cats), and/or Ursidae (e.g., giant panda).

Antibodies and Antigen Binding Fragments

The present disclosure provides anti-PD-1 antibodies and antigen-bindingfragments thereof. In general, antibodies (also called immunoglobulins)are made up of two classes of polypeptide chains, light chains and heavychains. A non-limiting antibody of the present disclosure can be anintact, four immunoglobulin chain antibody comprising two heavy chainsand two light chains. The heavy chain of the antibody can be of anyisotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype includingIgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chaincan be a kappa light chain or a lambda light chain.

An antibody can comprise two identical copies of a light chain and twoidentical copies of a heavy chain. The heavy chains, which each containone variable domain (or variable region, VH) and multiple constantdomains (or constant regions), bind to one another via disulfide bondingwithin their constant domains to form the “stem” of the antibody. Thelight chains, which each contain one variable domain (or variableregion, VL) and one constant domain (or constant region), each bind toone heavy chain via disulfide binding. The variable region of each lightchain is aligned with the variable region of the heavy chain to which itis bound. The variable regions of both the light chains and heavy chainscontain three hypervariable regions sandwiched between more conservedframework regions (FR).

These hypervariable regions, known as the complementary determiningregions (CDRs), form loops that comprise the principle antigen bindingsurface of the antibody. The four framework regions largely adopt abeta-sheet conformation and the CDRs form loops connecting, and in somecases forming part of, the beta-sheet structure. The CDRs in each chainare held in close proximity by the framework regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingregion.

Methods for identifying the CDR regions of an antibody by analyzing theamino acid sequence of the antibody are well known, and a number ofdefinitions of the CDRs are commonly used. The Kabat definition is basedon sequence variability, and the Chothia definition is based on thelocation of the structural loop regions. These methods and definitionsare described in, e.g., Martin, “Protein sequence and structure analysisof antibody variable domains,” Antibody engineering, Springer BerlinHeidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvementsto Kabat and structurally correct numbering of antibody variabledomains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, T. T. andKabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., MethodsEnzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16(October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998);Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko andBourne, BMC Structural Biology 7:64 (2007); each of which isincorporated herein by reference in its entirety.

The CDRs are important for recognizing an epitope of an antigen. As usedherein, an “epitope” is the smallest portion of a target moleculecapable of being specifically bound by the antigen binding domain of anantibody. The minimal size of an epitope may be about three, four, five,six, or seven amino acids, but these amino acids need not be in aconsecutive linear sequence of the antigen's primary structure, as theepitope may depend on an antigen's three-dimensional configuration basedon the antigen's secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule(e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses(IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in theirconstant region, particularly in their hinges and upper CH2 domains. Thesequences and differences of the IgG subclasses are known in the art,and are described, e.g., in Vidarsson, et al, “IgG subclasses andallotypes: from structure to effector functions.” Frontiers inimmunology 5 (2014); Irani, et al. “Molecular properties of human IgGsubclasses and their implications for designing therapeutic monoclonalantibodies against infectious diseases.” Molecular immunology 67.2(2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecularanalysis of structure, function and regulation. Elsevier, 2016; each ofwhich is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived fromany species (e.g., human, rodent, mouse, camelid, dog, cat, or giantpanda). Antibodies disclosed herein also include, but are not limitedto, polyclonal, monoclonal, monospecific, polyspecific antibodies, andchimeric antibodies that include an immunoglobulin binding domain fusedto another polypeptide. The term “antigen binding domain” or “antigenbinding fragment” is a portion of an antibody that retains specificbinding activity of the intact antibody, i.e., any portion of anantibody that is capable of specific binding to an epitope on the intactantibody's target molecule. It includes, e.g., Fab, Fab′, F(ab′)2, andvariants of these fragments. Thus, in some embodiments, an antibody oran antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, adAb, a bispecific antibody, a bispecific scFv, a diabody, a linearantibody, a single-chain antibody molecule, a multi-specific antibodyformed from antibody fragments, and any polypeptide that includes abinding domain which is, or is homologous to, an antibody bindingdomain. Non-limiting examples of antigen binding domains include, e.g.,the heavy chain and/or light chain CDRs of an intact antibody, the heavyand/or light chain variable regions of an intact antibody, full lengthheavy or light chains of an intact antibody, or an individual CDR fromeither the heavy chain or the light chain of an intact antibody.

In some embodiments, the antigen binding fragment can form a part of achimeric antigen receptor (CAR). In some embodiments, the chimericantigen receptor are fusions of single-chain variable fragments (scFv)as described herein, fused to CD3-zeta transmembrane- and endodomain. Insome embodiments, the chimeric antigen receptor also comprisesintracellular signaling domains from various costimulatory proteinreceptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimericantigen receptor comprises multiple signaling domains, e.g.,CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in oneaspect, the disclosure further provides cells (e.g., T cells) thatexpress the chimeric antigen receptors as described herein.

In some embodiments, the scFV has one heavy chain variable domain, andone light chain variable domain.

Anti-PD-1 Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereofthat specifically bind to PD-1 (e.g., canine PD-1). The antibodies andantigen-binding fragments described herein are capable of binding toPD-1 and can promote PD-1 signaling pathway thus increase immuneresponse. The disclosure provides e.g., mouse anti-PD-1 antibodies13-1B9 (“1B9”), 12-4A7 (“4A7”), and 12-1D8 (“1D8”), the chimericantibodies thereof, and the caninized antibodies thereof (e.g.,antibodies as shown in Table 1).

The CDR sequences for 1B9, and 1B9 derived antibodies (e.g., caninizedantibodies and felinized antibodies) include CDRs of the heavy chainvariable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variabledomain, SEQ ID NOs: 4-6 as defined by Kabat numbering. The CDRs can alsobe defined by Chothia system. Under the Chothia numbering, the CDRsequences of the heavy chain variable domain are set forth in SEQ IDNOs: 19-21 and CDR sequences of the light chain variable domain are setforth in SEQ ID NOs: 22-24.

Similarly, the CDR sequences for 4A7, and 4A7 derived antibodies includeCDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs ofthe light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabatnumbering. Under Chothia numbering, the CDR sequences of the heavy chainvariable domain are set forth in SEQ ID NOs: 25-27, and CDRs of thelight chain variable domain are set forth in SEQ ID NOs: 28-30.

The CDR sequences for 1D8, and 1D8 derived antibodies include CDRs ofthe heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of thelight chain variable domain, SEQ ID NOs: 16-18, as defined by Kabatnumbering. Under Chothia numbering, the CDR sequences of the heavy chainvariable domain are set forth in SEQ ID NOs: 31-33, and CDRs of thelight chain variable domain are set forth in SEQ ID NOs: 34-36.

The amino acid sequences for heavy chain variable regions and lightvariable regions of the caninized antibodies are also provided. As thereare different ways to caninize a mouse antibody (e.g., a sequence can bemodified with different amino acid substitutions), the heavy chain andthe light chain of an antibody can have more than one version ofcaninized sequences. The amino acid sequences for the heavy chainvariable regions of caninized 1B9 antibody are set forth in SEQ ID NOs:49-51. The amino acid sequences for the light chain variable regions ofcaninized 1B9 antibody are set forth in SEQ ID NOs: 52-54. Any of theseheavy chain variable region sequences (SEQ ID NO: 49-51) can be pairedwith any of these light chain variable region sequences (SEQ ID NO:52-54).

Similarly, the amino acid sequences for the heavy chain variable regionof caninized 1D8 antibody are set forth in SEQ ID NOs: 42-44. The aminoacid sequences for the light chain variable region of caninized 1D8antibody are set forth in SEQ ID NOs: 45-48. Any of these heavy chainvariable region sequences (SEQ ID NO: 42-44) can be paired with any ofthese light chain variable region sequences (SEQ ID NO: 45-48).

Caninization percentage means the percentage identity of the heavy chainor light chain variable region sequence as compared to a canine antibodysequences in International Immunogenetics Information System (IMGT)database. Atop hit means that the heavy chain or light chain variableregion sequence is closer to a particular species than to other species.In some embodiments, caninization percentage is greater than 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.A high caninization percentage often has various advantages, e.g., moresafe and more effective in dogs, more likely to be tolerated, and/orless likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-bindingfragments thereof described herein can also contain one, two, or threeheavy chain variable region CDRs selected from the group of SEQ ID NOs:1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs:25-27, and SEQ ID NOs: 31-33; and/or one, two, or three light chainvariable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ IDNOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs 28-30, andSEQ ID NOs: 34-36.

In some embodiments, the antibodies can have a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,3, wherein the CDR1 region comprises or consists of an amino acidsequence that is at least 80%, 85%, 90%, or 95% identical to a selectedVH CDR1 amino acid sequence, the CDR2 region comprises or consists of anamino acid sequence that is at least 80%, 85%, 90%, or 95% identical toa selected VH CDR2 amino acid sequence, and the CDR3 region comprises orconsists of an amino acid sequence that is at least 80%, 85%, 90%, or95% identical to a selected VH CDR3 amino acid sequence, and a lightchain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1region comprises or consists of an amino acid sequence that is at least80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acidsequence, the CDR2 region comprises or consists of an amino acidsequence that is at least 80%, 85%, 90%, or 95% identical to a selectedVL CDR2 amino acid sequence, and the CDR3 region comprises or consistsof an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected VHCDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3amino acid sequences are shown in FIG. 16 (Kabat CDR) and FIG. 17(Chothia CDR).

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 2 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 3 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 8 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 9 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 14 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 15 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 20 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 21 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 26 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 27 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 32 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 33 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 5 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 6 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 11 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 12 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 17 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 18 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 23 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 24 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 29 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 30 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 35 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 36 with zero, one or two amino acid insertions, deletions, orsubstitutions.

The insertions, deletions, and substitutions can be within the CDRsequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragmentsthereof that bind to PD-1 (e.g., canine PD-1). The antibodies orantigen-binding fragments thereof contain a heavy chain variable region(VH) comprising or consisting of an amino acid sequence that is at least80%, 85%, 90%, or 95% identical to a selected VH sequence, and a lightchain variable region (VL) comprising or consisting of an amino acidsequence that is at least 80%, 85%, 90%, or 95% identical to a selectedVL sequence. In some embodiments, the selected VH sequence is SEQ IDNOs: 49, 50, 51 or 59, and the selected VL sequence is SEQ ID NOs: 52,53, 54, or 60. In some embodiments, the selected VH sequence is SEQ IDNOs: 42, 43, 44, or 57 and the selected VL sequence is SEQ ID NOs: 45,46, 47, 48, or 58. In some embodiments, the selected VH sequence is SEQID NO: 55, and the selected VL sequence is SEQ ID NO: 56.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90%, 95%, or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. For purposes of the present disclosure, the comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The disclosure also provides nucleic acid comprising a polynucleotideencoding a polypeptide comprising an immunoglobulin heavy chain or animmunoglobulin light chain. The immunoglobulin heavy chain orimmunoglobulin light chain comprises CDRs as shown in FIG. 16 or FIG.17, or have sequences as shown in FIGS. 19-21. When the polypeptides arepaired with corresponding polypeptide (e.g., a corresponding heavy chainvariable region or a corresponding light chain variable region), thepaired polypeptides bind to PD-1 (e.g., canine PD-1).

The anti-PD-1 antibodies and antigen-binding fragments can also beantibody variants (including derivatives and conjugates) of antibodiesor antibody fragments and multi-specific (e.g., bi-specific) antibodiesor antibody fragments. Additional antibodies provided herein arepolyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific),human antibodies, canine antibodies, feline antibodies, pandaantibodies, chimeric antibodies (e.g., human-mouse chimera, canine-mousechimera), single-chain antibodies, intracellularly-made antibodies(i.e., intrabodies), and antigen-binding fragments thereof. Theantibodies or antigen-binding fragments thereof can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody orantigen-binding fragment thereof is an IgG antibody or antigen-bindingfragment thereof (e.g., canine, feline, or panda antibody).

In some embodiments, the antibody described herein is a canine orcaninized IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4). For example,the antibody can have a canine IgG constant regions, including CL, CH1,CH2, and/or CH3. In some embodiments, the antibody is a feline orfelinized IgG antibody. For example, the antibody can have a feline IgGconstant regions, including CL, CH1, CH2, and/or CH3. In someembodiments, the antibody is a panda IgG antibody. In some cases, it canhave constant regions (e.g., CL, CH1, CH2, and/or CH3) of a panda IgGantibody.

Fragments of antibodies are suitable for use in the methods provided solong as they retain the desired affinity and specificity of thefull-length antibody. Thus, a fragment of an antibody that binds to PD-1will retain an ability to bind to PD-1. An Fv fragment is an antibodyfragment which contains a complete antigen recognition and binding site.This region consists of a dimer of one heavy and one light chainvariable domain in tight association, which can be covalent in nature,for example in scFv. It is in this configuration that the three CDRs ofeach variable domain interact to define an antigen binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs or a subsetthereof confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) can have the ability to recognize and bindantigen, although usually at a lower affinity than the entire bindingsite.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VLdomains (or regions) of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the scFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains, whichenables the scFv to form the desired structure for antigen binding.

The Fab fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)2 antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

Diabodies are small antibody fragments with two antigen-binding sites.The fragments comprise a VH connected to a VL in the same polypeptidechain (VH and VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites.

Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1)which, together with complementary light chain polypeptides, form a pairof antigen binding regions. Linear antibodies can be bispecific ormonospecific.

Antibodies and antibody fragments of the present disclosure can bemodified in the Fc region to provide desired effector functions or serumhalf-life.

Multimerization of antibodies may be accomplished through naturalaggregation of antibodies or through chemical or recombinant linkingtechniques known in the art. For example, some percentage of purifiedantibody preparations (e.g., purified IgG1 molecules) spontaneously formprotein aggregates containing antibody homodimers and other higher-orderantibody multimers.

Alternatively, antibody homodimers may be formed through chemicallinkage techniques known in the art. For example, heterobifunctionalcrosslinking agents including, but not limited to SMCC (succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidylS-acethylthio-acetate) can be used to form antibody multimers. Anexemplary protocol for the formation of antibody homodimers is describedin Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997).Antibody homodimers can be converted to Fab′2 homodimers throughdigestion with pepsin. Another way to form antibody homodimers isthrough the use of the autophilic T15 peptide described in Zhao et al.(J. Immunol. 25:396-404, 2002).

In some embodiments, the multi-specific antibody is a bi-specificantibody. Bi-specific antibodies can be made by engineering theinterface between a pair of antibody molecules to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. For example, the interface can contain at least a part of theCH3 domain of an antibody constant domain. In this method, one or moresmall amino acid side chains from the interface of the first antibodymolecule are replaced with larger side chains (e.g., tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g., alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers. This method is described, e.g., in WO 96/27011, which isincorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin and the other to biotin. Heteroconjugateantibodies can also be made using any convenient cross-linking methods.Suitable cross-linking agents and cross-linking techniques are wellknown in the art and are disclosed in U.S. Pat. No. 4,676,980, which isincorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragmentsare also known in the art. For example, bi-specific antibodies can beprepared using chemical linkage. Brennan et al. (Science 229:81, 1985)describes a procedure where intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′ TNB derivatives isthen reconverted to the Fab′ thiol by reduction with mercaptoethylamine,and is mixed with an equimolar amount of another Fab′ TNB derivative toform the bi-specific antibody.

Any of the antibodies or antigen-binding fragments described herein maybe conjugated to a stabilizing molecule (e.g., a molecule that increasesthe half-life of the antibody or antigen-binding fragment thereof in asubject or in solution). Non-limiting examples of stabilizing moleculesinclude: a polymer (e.g., a polyethylene glycol) or a protein (e.g.,serum albumin, such as canine serum albumin). The conjugation of astabilizing molecule can increase the half-life or extend the biologicalactivity of an antibody or an antigen-binding fragment in vitro (e.g.,in tissue culture or when stored as a pharmaceutical composition) or invivo.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein can be conjugated to a therapeutic agent. Theantibody-drug conjugate comprising the antibody or antigen-bindingfragment thereof can covalently or non-covalently bind to a therapeuticagent. In some embodiments, the therapeutic agent is a cytotoxic orcytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoidssuch as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

Antibody Characteristics

The antibodies or antigen-binding fragments thereof described herein canblock the binding between PD-1 and PD-L1 and/or the binding between PD-1and PD-L2.

In some embodiments, by binding to PD-1, the antibody can inhibit PD-1signaling pathway and upregulates the immune response. Thus, in someembodiments, the antibodies or antigen-binding fragments thereof asdescribed herein are PD-1 antagonist. In some embodiments, theantibodies or antigen-binding fragments thereof are PD-1 agonist. Insome embodiments, the antibodies or antigen-binding fragments thereofcan block the binding between canine PD-1 and canine PD-L1, feline PD-1and feline PD-L1, and/or panda PD-1 and panda PD-L1.

In some embodiments, the antibodies or antigen-binding fragments thereofas described herein can increase immune response, activity or number ofT cells (e.g., CD8+ and/or CD4+ cells) by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or20 folds. In some embodiments, the antibodies or antigen-bindingfragments thereof as described herein can decrease the activity ornumber of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some implementations, the antibody (or antigen-binding fragmentsthereof) specifically binds to PD-1 (e.g., canine PD-1, feline PD-1,panda PD-1, monkey PD-1, mouse PD-1, and/or chimeric PD-1) with adissociation rate (koff) of less than 0.1 s⁻¹, less than 0.01 s⁻¹, lessthan 0.001 s⁻¹, less than 0.0001 s⁻¹, or less than 0.00001 s⁻¹. In someembodiments, the dissociation rate (koff) is greater than 0.01 s⁻¹,greater than 0.001 s⁻¹, greater than 0.0001 s⁻¹, greater than 0.00001s⁻¹, or greater than 0.000001 s⁻¹.

In some embodiments, kinetic association rates (kon) is greater than1×10²/Ms, greater than 1×10³/Ms, greater than 1×10⁴/Ms, greater than1×10⁵/Ms, or greater than 1×10⁶/Ms. In some embodiments, kineticassociation rates (kon) is less than 1×10⁵/Ms, less than 1×10⁶/Ms, orless than 1×10⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rateconstants (KD=koff/kon). In some embodiments, KD is less than 1×10⁻⁶ M,less than 1×10⁻⁷M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, or less than1×10⁻¹⁰ M. In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM,15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.In some embodiments, KD is greater than 1×10⁻⁷M, greater than 1×10⁻⁸ M,greater than 1×10⁻⁹ M, greater than 1×10⁻¹⁰ M, greater than 1×10⁻¹¹ M,or greater than 1×10⁻¹² M. In some embodiments, the antibody binds tocanine PD-1 with KD less than or equal to about 6 nM, 5 nM, 4 nM, 3 nM,2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2nM, or 0.1 nM.

General techniques for measuring the affinity of an antibody for anantigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR).In some embodiments, the antibody binds to canine PD-1 (SEQ ID NO: 41),feline PD-1 (SEQ ID NO: 62), panda PD-1 (SEQ ID NO: 31), human PD-1 (SEQID NO: 37), monkey PD-1 (e.g., rhesus macaque PD-1, SEQ ID NO: 39),chimeric PD-1 (SEQ ID NO: 40), and/or mouse PD-1 (SEQ ID NO: 38). Insome embodiments, the antibody does not bind to canine PD-1 (SEQ ID NO:41), feline PD-1 (SEQ ID NO: 62), panda PD-1 (SEQ ID NO: 31), human PD-1(SEQ ID NO: 37), monkey PD-1 (e.g., rhesus macaque PD-1, SEQ ID NO: 39),chimeric PD-1 (SEQ ID NO: 40), and/or mouse PD-1 (SEQ ID NO: 38).

In some embodiments, the EC50 for the binding activity with canine PD-1,feline PD-1, or panda PD-1 is less than 500 nM, 400 nM, 300 nM, 200 nM,100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In someembodiments, the EC50 for the binding activity with canine PD-1, felinePD-1, or panda PD-1 is greater than 100 nM, 90 nM, 80 nM, 70 nM, 60 nM,50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3nM, 2 nM, or 1 nM. As used herein, the term “EC50” refers to theconcentration for inducing half maximal effective response. At the EC50concentration, an agent (e.g., a drug, antibody or toxicant) induces aresponse halfway between the baseline and maximum after a specifiedexposure time. “EC50” for the binding activity refers to theconcentration of an agent (e.g., a drug, antibody or toxicant) whichprovides a binding activity halfway between the baseline and maximumbinding activity.

In some embodiments, thermal stabilities are determined. The antibodiesor antigen binding fragments as described herein can have a Tm greaterthan 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,or 95° C.

As IgG can be described as a multi-domain protein, the melting curvesometimes shows two transitions, with a first denaturation temperature,Tm D1, and a second denaturation temperature Tm D2. The presence ofthese two peaks often indicate the denaturation of the Fc domains (TmD1) and Fab domains (Tm D2), respectively. When there are two peaks, Tmusually refers to Tm D2. Thus, in some embodiments, the antibodies orantigen binding fragments as described herein has a Tm D1 greater than60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or95° C. In some embodiments, the antibodies or antigen binding fragmentsas described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, the antibody has a tumor growth inhibitionpercentage (TGI %) that is greater than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, or 200%. In some embodiments, the antibody has a tumor growthinhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI % canbe determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after thetreatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 monthsafter the treatment starts. As used herein, the tumor growth inhibitionpercentage (TGI %) is calculated using the following formula:

TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100

Ti is the average tumor volume in the treatment group on day i. T0 isthe average tumor volume in the treatment group on day zero. Vi is theaverage tumor volume in the control group on day i. V0 is the averagetumor volume in the control group on day zero.

In some embodiments, the antibodies or antigen-binding fragments thereofas described herein are PD-1 antagonist. In some embodiments, theantibodies or antigen binding fragments decrease PD-1 signaltransduction in a target cell that expresses PD-1.

In some embodiments, the antibodies or antigen binding fragments enhanceCD4+ effector T cell function, for example, by increasing CD4+ effectorT cell proliferation and/or increasing gamma interferon production bythe CD4+ effector T cell (e.g., as compared to proliferation and/orcytokine production prior to treatment with the antibodies or antigenbinding fragments). In some embodiments, the cytokine is gammainterferon. In some embodiments, the antibodies or antigen bindingfragments increase number of intratumoral (infiltrating) CD4+ effector Tcells (e.g., total number of CD4+ effector T cells, or e.g., percentageof CD4+ cells in CD45+ cells), e.g., as compared to number ofintratumoral (infiltrating) CD4+ T cells prior to treatment withantibodies or antigen binding fragments. In some embodiments, theantibodies or antigen binding fragments increase number of intratumoral(infiltrating) CD4+ effector T cells that express gamma interferon(e.g., total gamma interferon expressing CD4+ cells, or e.g., percentageof gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., ascompared to number of intratumoral (infiltrating) CD4+ T cells thatexpress gamma interferon prior to treatment.

In some embodiments, the antibodies or antigen binding fragmentsincrease number of intratumoral (infiltrating) CD8+ effector T cells(e.g., total number of CD8+ effector T cells, or e.g., percentage ofCD8+ in CD45+ cells), e.g., as compared to number of intratumoral(infiltrating) CD8+T effector cells prior to treatment. In someembodiments, the antibodies or antigen binding fragments increase numberof intratumoral (infiltrating) CD8+ effector T cells that express gammainterferon (e.g., percentage of CD8+ cells that express gamma interferonin total CD8+ cells), e.g., compared to number of intratumoral(infiltrating) CD8+ T cells that express gamma interferon prior totreatment with anti-PD-1 antibody (e.g., anti-canine PD-1 antibody).

In some embodiments, the antibodies or antigen binding fragments enhancememory T cell function, for example by increasing memory T cellproliferation and/or increasing cytokine (e.g., gamma interferon)production by the memory cell.

In some embodiments, the antibodies or antigen binding fragments inhibitTreg function, for example, by decreasing Treg suppression of effector Tcell function (e.g., effector T cell proliferation and/or effector Tcell cytokine secretion). In some embodiments, the effector T cell is aCD4+ effector T cell. In some embodiments, the antibodies or antigenbinding fragments reduce the number of intratumoral (infiltrating) Treg(e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+cells).

In some embodiments, the antibodies or antigen binding fragments aredepleting anti-PD-1 antibody (e.g., depletes cells that express caninePD-1). In some embodiments, the antibodies or antigen binding fragmentsdeplete cells that express PD-1 (e.g., canine PD-1) in vitro. In someembodiments, the PD-1 expressing cells are CD4+ effector T cells, orTreg cells. In some embodiments, depleting is by ADCC and/orphagocytosis.

In some embodiments, the antibodies or antigen binding fragments have afunctional Fc region. In some embodiments, effector function of afunctional Fc region is antibody-dependent cell-mediated cytotoxicity(ADCC). In some embodiments, effector function of a functional Fc regionis phagocytosis. In some embodiments, effector function of a functionalFc region is ADCC and phagocytosis. In some embodiments, the Fc regionis of canine IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

In some embodiments, the antibodies or antigen binding fragments do notinduce apoptosis in PD-1-expressing cells (e.g., Treg).

In some embodiments, the antibodies or antigen binding fragments do nothave a functional Fc region. For example, the antibodies or antigenbinding fragments are Fab, Fab′, F(ab′)2, and Fv fragments.

Methods of Making Anti-PD-1 Antibodies

An isolated fragment of PD-1 (e.g., canine PD-1, feline PD-1, or pandaPD-1) can be used as an immunogen to generate antibodies using standardtechniques for polyclonal and monoclonal antibody preparation.Polyclonal antibodies can be raised in animals by multiple injections(e.g., subcutaneous or intraperitoneal injections) of an antigenicpeptide or protein. In some embodiments, the antigenic peptide orprotein is injected with at least one adjuvant. In some embodiments, theantigenic peptide or protein can be conjugated to an agent that isimmunogenic in the species to be immunized. Animals can be injected withthe antigenic peptide or protein more than one time (e.g., twice, threetimes, or four times).

The full-length polypeptide or protein can be used or, alternatively,antigenic peptide fragments thereof can be used as immunogens. Theantigenic peptide of a protein comprises at least 8 (e.g., at least 10,15, 20, or 30) amino acid residues of the amino acid sequence of PD-1and encompasses an epitope of the protein such that an antibody raisedagainst the peptide forms a specific immune complex with the protein. Asdescribed above, the full length sequence of canine PD-1 is known in theart (SEQ ID NO: 41).

An immunogen typically is used to prepare antibodies by immunizing asuitable subject (e.g., a mouse, or a transgenic animal). An appropriateimmunogenic preparation can contain, for example, arecombinantly-expressed or a chemically-synthesized polypeptide (e.g., afragment of canine PD-1). The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or a similarimmunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a PD-1 polypeptide, or an antigenic peptidethereof (e.g., part of PD-1) as an immunogen. The antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme-linked immunosorbent assay (ELISA) using theimmobilized PD-1 polypeptide or peptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein A of proteinG chromatography to obtain the IgG fraction. At an appropriate timeafter immunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler et al. (Nature256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al.,Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985), or trioma techniques. The technology for producing hybridomas iswell known (see, generally, Current Protocols in Immunology, 1994,Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.).Hybridoma cells producing a monoclonal antibody are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide or epitope of interest, e.g., using a standard ELISAassay.

Variants of the antibodies or antigen-binding fragments described hereincan be prepared by introducing appropriate nucleotide changes into theDNA encoding a canine, feline, caninized, felinized, or chimericantibody, or antigen-binding fragment thereof described herein, or bypeptide synthesis. Such variants include, for example, deletions,insertions, or substitutions of residues within the amino acidssequences that make-up the antigen-binding site of the antibody or anantigen-binding domain. In a population of such variants, someantibodies or antigen-binding fragments will have increased affinity forthe target protein, e.g., PD-1. Any combination of deletions,insertions, and/or combinations can be made to arrive at an antibody orantigen-binding fragment thereof that has increased binding affinity forthe target. The amino acid changes introduced into the antibody orantigen-binding fragment can also alter or introduce newpost-translational modifications into the antibody or antigen-bindingfragment, such as changing (e.g., increasing or decreasing) the numberof glycosylation sites, changing the type of glycosylation site (e.g.,changing the amino acid sequence such that a different sugar is attachedby enzymes present in a cell), or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal,including mammals. Non-limiting examples of native antibodies includeantibodies derived from humans, dogs, cats, pandas, primates, e.g.,monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels andllamas), chicken, goats, and rodents (e.g., rats, mice, hamsters andrabbits), including transgenic rodents genetically engineered to producehuman, canine, or feline antibodies.

The present disclosure also further provides cells and cell linesexpressing antibodies described herein. Representative host cellsinclude e.g., bacterial, yeast, mammalian and human cells, such as CHOcells, HEK-293 cells, HeLa cells, CV-1 cells, and COS cells. Methods forgenerating a stable cell line following transformation of a heterologousconstruct into a host cell are known in the art. Representativenon-mammalian host cells include insect cells (Potter et al. (1993) Int.Rev. Immunol. 10(2-3):103-112). Antibodies can also be produced intransgenic animals (Houdebine (2002) Curr. Opin. Biotechnol.13(6):625-629) and transgenic plants (Schillberg et al. (2003) Cell Mol.Life Sci. 60(3):433-45).

Ordinarily, amino acid sequence variants of the canine, caninized,feline, felinized or chimeric anti-PD-1 antibody will contain an aminoacid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% percent identity with a sequence present in the light or heavy chainof the original antibody.

Identity or homology with respect to an original sequence is usually thepercentage of amino acid residues present within the candidate sequencethat are identical with a sequence present within the canine, caninized,feline, felinized, or chimeric anti-PD-1 antibody or fragment, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity.

Additional modifications to the anti-PD-1 antibodies or antigen-bindingfragments can be made. For example, a cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have any increased half-life in vitro and/or in vivo. Homodimericantibodies with increased half-life in vitro and/or in vivo can also beprepared using heterobifunctional cross-linkers as described, forexample, in Wolff et al. (Cancer Res. 53:2560-2565, 1993).Alternatively, an antibody can be engineered which has dual Fc regions(see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230,1989).

In some embodiments, a covalent modification can be made to theanti-PD-1 antibody or antigen-binding fragment thereof. These covalentmodifications can be made by chemical or enzymatic synthesis, or byenzymatic or chemical cleavage. Other types of covalent modifications ofthe antibody or antibody fragment are introduced into the molecule byreacting targeted amino acid residues of the antibody or fragment withan organic derivatization agent that is capable of reacting withselected side chains or the N- or C-terminal residues.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e.g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry,as described in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues; or position 314 in Kabat numbering);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. In some embodiments, to reduce glycanheterogeneity, the Fc region of the antibody can be further engineeredto replace the Asparagine at position 297 with Alanine (N297A).

In some embodiments, to facilitate production efficiency by avoidingFab-arm exchange, the Fc region of the antibodies was further engineeredto replace the serine at position 228 (EU numbering) of IgG4 withproline (S228P). A detailed description regarding S228 mutation isdescribed, e.g., in Silva et al. “The S228P mutation prevents in vivoand in vitro IgG4 Fab-arm exchange as demonstrated using a combinationof novel quantitative immunoassays and physiological matrixpreparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469,which is incorporated by reference in its entirety.

Speciation of Antibodies

The generation of anti-drug antibodies (ADAs) can been associated withloss of efficacy for biotherapeutic protein including monoclonalantibodies. Comprehensive evaluation of the literature has shown thatspeciation of monoclonal antibodies can reduce the propensity for mAbsto be immunogenic. To help mitigate risks associated with ADA formationfor the mouse anti PD-1 monoclonal antibodies provided herein, aspeciation strategy (e.g., caninization or felinization) can beemployed. This strategy can be based on identifying the most appropriatecanine or feline germline antibody sequence for CDR grafting. Followingextensive analysis of all available canine germline sequences for boththe heavy and light chain, germline candidates can be selected based ontheir homology to the mouse mAbs, and the CDRs from the mouse progenitormAbs can be used to replace native canine or feline CDRs. The objectiveis to retain high affinity and cell-based activity using fully canine orfeline frameworks to minimize the potential of immunogenicity in vivo.Caninized or felinized mAbs are expressed and characterized for theirability to bind canine or feline PD-1 via Western blotting.

Caninized or felinized antibodies include antibodies having variable andconstant regions derived from (or having the same amino acid sequence asthose derived from) canine or feline germline immunoglobulin sequences.In some cases, caninized or felinized antibodies may include amino acidresidues not encoded by canine or feline germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs.

A caninized or felinized antibody, typically has a canine or felineframework (FR) grafted with CDRs of an antibody derived from anotherspecies (e.g., mouse). Thus, a caninized or felinized antibody has oneor more amino acid sequence introduced into it from a source other thana canine or a feline mammal. These amino acid residues are oftenreferred to as “import” residues, which are typically taken from an“import” variable domain. Caninization or felinization can beessentially performed by e.g., substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a canine or feline antibody. Thesemethods are described in e.g., Singer, et al. “Generation of a canineanti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancerpatients.” Molecular cancer therapeutics (2014); Maekawa, et al. “Acanine chimeric monoclonal antibody targeting PD-L1 and its clinicalefficacy in canine oral malignant melanoma or undifferentiated sarcoma.”Scientific reports 7.1 (2017): 8951; US20170158756A1; each of which isincorporated by reference herein in its entirety. Accordingly,“caninized” antibodies are chimeric antibodies wherein substantiallyless than an intact canine V domain has been substituted by thecorresponding sequence from a non-canine species, and “felinized”antibodies are chimeric antibodies wherein substantially less than anintact feline V domain has been substituted by the correspondingsequence from a non-feline species. In practice, these antibodies aretypically mouse antibodies in which some CDR residues and some FRresidues are substituted by residues from analogous sites in canine orfeline antibodies.

Framework residues are those residues of antibody variable regions otherthan hypervariable or CDR residues. Framework residues may be derivedfrom a naturally occurring canine or feline antibody, such as a canineor feline framework that is substantially similar to a framework regionof the antibody described herein. Artificial framework sequences thatrepresent a consensus among individual sequences may also be used. Whenselecting a framework region for caninization or felinization, sequencesthat are widely represented in canines or felines may be preferred overless populous sequences. Additional mutations of the framework acceptorsequences may be made to restore murine residues believed to be involvedin antigen contacts and/or residues involved in the structural integrityof the antigen-binding site, or to improve antibody expression. When theresidues of the framework regions in the VH and VL are derived, at leastin part, from canine or feline antibody sequences, variable regions arealso described as caninized or felinized (e.g., a caninized or felinizedlight or heavy chain variable region).

Grafting of CDRs is performed by replacing one or more CDRs of anacceptor antibody (e.g., a caninized antibody or other antibodycomprising desired framework residues) with CDRs of a donor antibody(e.g., a non-canine antibody). Acceptor antibodies can be selected basedon similarity of framework residues between a candidate acceptorantibody and a donor antibody. By using the same methods, CDRs can begrafted to an antibody of Ailuropoda melanoleuca (giant panda).

The sequence for the constant regions for canine and feline IgG areknown in the art. SEQ ID NO: 63 shows the amino acid sequence for thecanine immunoglobulin gamma heavy chain A (IgG1) constant region(GenBank accession no. AAL35301.1). SEQ ID NO: 64 shows the amino acidsequence for the canine immunoglobulin gamma heavy chain B (IgG2)constant region (GenBank accession no. AAL35302.1). SEQ ID NO: 68 showsthe amino acid sequence for the canine immunoglobulin gamma heavy chainC (IgG3) constant region (GenBank accession no. AAL35303.1). SEQ ID NO:69 shows the amino acid sequence for the canine immunoglobulin gammaheavy chain D (IgG4) constant region (GenBank accession no. AAL35304.1).

SEQ ID NO: 65 shows the amino acid sequence for the canine kappa chain(GenBank accession no. XP_532962.3). SEQ ID NO: 70 and 71 show the aminoacid sequence for the canine IgG light chain constant regions.

SEQ ID NO: 66 is the amino acid sequence for the feline heavy chainconstant region (GenBank: BAA32229.1). SEQ ID NO: 67 is the amino acidsequence for the feline kappa chain constant region (GenBank:AAF09245.1).

It is further important that antibodies be caninized or felinized withretention of high specificity and affinity for the antigen and otherfavorable biological properties. To achieve this goal, caninized orfelinized antibodies can be prepared by a process of analysis of theparental sequences and various conceptual caninized or felinizedproducts using three-dimensional models of the parental and caninized orfelinized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved.

In some cases, back mutation of selected residues in the variable regionis used to enhance presentation of the CDRs. Designing antibodies thatminimize immunogenic reaction in a subject to non-native sequences inthe antibody, while at the same time preserving antigen binding regionsof the antibody sufficiently to maintain efficacy, has provenchallenging. As used herein, the term “back mutation” refers to aprocess in which some or all of the somatically mutated amino acids of acanine or feline antibody are replaced with the corresponding germlineresidues from a homologous germline antibody sequence. The heavy andlight chain sequences of the canine or feline antibody are alignedseparately with the germline sequences to identify the sequences withthe highest homology. Differences in the canine or feline antibody arereturned to the germline sequence by mutating defined nucleotidepositions encoding such different amino acid.

The role of each amino acid thus identified as candidate for backmutation should be investigated for a direct or indirect role in antigenbinding and any amino acid found after mutation to affect any desirablecharacteristic of the antibody should not be included in the finalantibody; as an example, activity enhancing amino acids identified bythe selective mutagenesis approach will not be subject to back mutation.To minimize the number of amino acids subject to back mutation thoseamino acid positions found to be different from the closest germlinesequence but identical to the corresponding amino acid in a secondgermline sequence can remain, provided that the second germline sequenceis identical and colinear to the sequence of the antibody. Back mutationof selected target framework residues to the corresponding donorresidues might be required to restore and or improved affinity.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., anexpression vectors) that include an isolated polynucleotide disclosedherein (e.g., a polynucleotide that encodes a polypeptide disclosedherein), host cells into which are introduced the recombinant vectors(i.e., such that the host cells contain the polynucleotide and/or avector comprising the polynucleotide), and the production of recombinantantibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one ormore polynucleotide(s) of interest to a host cell when the vector isintroduced to the host cell. An “expression vector” is capable ofdelivering and expressing the one or more polynucleotide(s) of interestas an encoded polypeptide in a host cell into which the expressionvector has been introduced. Thus, in an expression vector, thepolynucleotide of interest is positioned for expression in the vector bybeing operably linked with regulatory elements such as a promoter,enhancer, and/or a poly-A tail, either within the vector or in thegenome of the host cell at or near or flanking the integration site ofthe polynucleotide of interest such that the polynucleotide of interestwill be translated in the host cell introduced with the expressionvector.

A vector can be introduced into the host cell by methods known in theart, e.g., electroporation, chemical transfection (e.g., DEAE-dextran),transformation, transfection, and infection and/or transduction (e.g.,with recombinant virus). Thus, non-limiting examples of vectors includeviral vectors (which can be used to generate recombinant virus), nakedDNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expressionvectors associated with cationic condensing agents.

In some embodiments, a polynucleotide disclosed herein (e.g., apolynucleotide that encodes a polypeptide disclosed herein) isintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus, or may use areplication defective virus. In the latter case, viral propagationgenerally will occur only in complementing virus packaging cells.Suitable systems are disclosed, for example, in Fisher-Hoch et al.,1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann.N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S.Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat.No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science,252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219;Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502;Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993,Cir. Res., 73:1202-1207. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692.The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding orpolypeptide-encoding polynucleotide disclosed herein can be operativelylinked to an appropriate promoter (e.g., a heterologous promoter), suchas the phage lambda PL promoter, the E. coli lac, trp and tac promoters,the SV40 early and late promoters and promoters of retroviral LTRs, toname a few. Other suitable promoters are known to the skilled artisan.The expression constructs can further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs may include a translationinitiating at the beginning and a termination codon (UAA, UGA, or UAG)appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectablemarker. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracycline or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces, andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells.Appropriate culture mediums and conditions for the host cells describedherein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Non-limiting bacterial promoters suitable for use include the E. colilacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, thelambda PR and PL promoters and the trp promoter. Suitable eukaryoticpromoters include the CMV immediate early promoter, the HSV thymidinekinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous sarcoma virus (RSV), andmetallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986), which is incorporated herein by reference in itsentirety.

Transcription of DNA encoding an antibody of the present disclosure byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at base pairs 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a modified form,such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag,and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties can be added to the polypeptide tofacilitate purification. Such regions can be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Methods of Treatment

The antibodies or antibody or antigen-binding fragments thereof of thepresent disclosure can be used for various therapeutic purposes. In oneaspect, the disclosure provides methods for treating a cancer in asubject, methods of reducing the rate of the increase of volume of atumor in a subject over time, methods of reducing the risk of developinga metastasis, or methods of reducing the risk of developing anadditional metastasis in a subject. In some embodiments, the treatmentcan halt, slow, retard, or inhibit progression of a cancer. In someembodiments, the treatment can result in the reduction of in the number,severity, and/or duration of one or more symptoms of the cancer in asubject.

In one aspect, the disclosure features methods that includeadministering a therapeutically effective amount of an antibody orantigen-binding fragment thereof disclosed herein to a subject in needthereof (e.g., a subject having, or identified or diagnosed as having, acancer), e.g., breast cancer (e.g., triple-negative breast cancer),carcinoid cancer, cervical cancer, endometrial cancer, glioma, head andneck cancer, liver cancer, lung cancer, small cell lung cancer,lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer,renal cancer, colorectal cancer, gastric cancer, testicular cancer,thyroid cancer, bladder cancer, urethral cancer, or hematologicmalignancy. In some embodiments, the cancer is unresectable melanoma ormetastatic melanoma, non-small cell lung carcinoma (NSCLC), small celllung cancer (SCLC), bladder cancer, or metastatic hormone-refractoryprostate cancer. In some embodiments, the subject has a solid tumor. Insome embodiments, the cancer is squamous cell carcinoma of the head andneck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer(TNBC), or colorectal carcinoma. In some embodiments, the subject hasHodgkin's lymphoma. In some embodiments, the subject has triple-negativebreast cancer (TNBC), gastric cancer, urothelial cancer, Merkel-cellcarcinoma, or head and neck cancer.

In some embodiments, the compositions and methods disclosed herein canbe used for treatment of patients at risk for a cancer. Patients withcancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosagesufficient to effect beneficial or desired results including halting,slowing, retarding, or inhibiting progression of a disease, e.g., acancer. An effective amount will vary depending upon, e.g., an age and abody weight of a subject to which the antibody, antigen bindingfragment, antibody-encoding polynucleotide, vector comprising thepolynucleotide, and/or compositions thereof is to be administered, aseverity of symptoms and a route of administration, and thusadministration can be determined on an individual basis.

An effective amount can be administered in one or more administrations.By way of example, an effective amount of an antibody or an antigenbinding fragment is an amount sufficient to ameliorate, stop, stabilize,reverse, inhibit, slow and/or delay progression of a cancer in a patientor is an amount sufficient to ameliorate, stop, stabilize, reverse, slowand/or delay proliferation of a cell (e.g., a biopsied cell, any of thecancer cells described herein, or cell line (e.g., a cancer cell line))in vitro. As is understood in the art, an effective amount of anantibody or antigen binding fragment may vary, depending on, inter alia,patient history as well as other factors such as the type (and/ordosage) of antibody used.

Effective amounts and schedules for administering the antibodies,antibody-encoding polynucleotides, and/or compositions disclosed hereinmay be determined empirically, and making such determinations is withinthe skill in the art. Those skilled in the art will understand that thedosage that must be administered will vary depending on, for example,the mammal that will receive the antibodies, antibody-encodingpolynucleotides, and/or compositions disclosed herein, the route ofadministration, the particular type of antibodies, antibody-encodingpolynucleotides, antigen binding fragments, and/or compositionsdisclosed herein used and other drugs being administered to the mammal.Guidance in selecting appropriate doses for antibody or antigen bindingfragment can be found in the literature on therapeutic uses ofantibodies and antigen binding fragments, e.g., Handbook of MonoclonalAntibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J.,1985, ch. 22 and pp. 303-357.

A typical daily dosage of an effective amount of an antibody is 0.01mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments,the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage isabout 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody,antigen-binding fragment thereof, or pharmaceutical composition (e.g.,any of the antibodies, antigen-binding fragments, or pharmaceuticalcompositions described herein) and, optionally, at least one additionaltherapeutic agent can be administered to the subject at least once aweek (e.g., once a week, twice a week, three times a week, four times aweek, once a day, twice a day, or three times a day). In someembodiments, at least two different antibodies and/or antigen-bindingfragments are administered in the same composition (e.g., a liquidcomposition). In some embodiments, at least one antibody orantigen-binding fragment and at least one additional therapeutic agentare administered in the same composition (e.g., a liquid composition).In some embodiments, the at least one antibody or antigen-bindingfragment and the at least one additional therapeutic agent areadministered in two different compositions (e.g., a liquid compositioncontaining at least one antibody or antigen-binding fragment and a solidoral composition containing at least one additional therapeutic agent).In some embodiments, the at least one additional therapeutic agent isadministered as a pill, tablet, or capsule. In some embodiments, the atleast one additional therapeutic agent is administered in asustained-release oral formulation.

In some embodiments, the one or more additional therapeutic agents canbe administered to the subject prior to, or after administering the atleast one antibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein). In someembodiments, the one or more additional therapeutic agents and the atleast one antibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein) areadministered to the subject such that there is an overlap in thebioactive period of the one or more additional therapeutic agents andthe at least one antibody or antigen-binding fragment (e.g., any of theantibodies or antigen-binding fragments described herein) in thesubject.

In some embodiments, the subject can be administered the at least oneantibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein) over anextended period of time (e.g., over a period of at least 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1year, 2 years, 3 years, 4 years, or 5 years). A skilled medicalprofessional may determine the length of the treatment period using anyof the methods described herein for diagnosing or following theeffectiveness of treatment (e.g., the observation of at least onesymptom of cancer). As described herein, a skilled medical professionalcan also change the identity and number (e.g., increase or decrease) ofantibodies or antigen-binding antibody fragments (and/or one or moreadditional therapeutic agents) administered to the subject and can alsoadjust (e.g., increase or decrease) the dosage or frequency ofadministration of at least one antibody or antigen-binding antibodyfragment (and/or one or more additional therapeutic agents) to thesubject based on an assessment of the effectiveness of the treatment(e.g., using any of the methods described herein and known in the art).

In some embodiments, one or more additional therapeutic agents can beadministered to the subject. The additional therapeutic agent cancomprise one or more inhibitors selected from the group consisting of aninhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, aninhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, aninhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of aphosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, aninhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton'styrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1(IDH1) and/or Isocitrate dehydrogenase 2 (IDH2). In some embodiments,the additional therapeutic agent is an inhibitor of indoleamine2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

In some embodiments, the additional therapeutic agent can comprise oneor more inhibitors selected from the group consisting of an inhibitor ofHER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2,an inhibitor of CHK1, an inhibitor of activated hedgehog signalingpathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting ofTrabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib,Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin,Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib,Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, anHsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine,Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine,lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, andenzastaurin.

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting of anadjuvant, a TAR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1,an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17antagonist, an HVEM antagonist, an ICOS agonist, a treatment targetingCX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, atreatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and aSelectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin,pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to thesubject.

In some embodiments, the additional therapeutic agent is an anti-OX40antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4antibody, or an anti-GITR antibody.

Methods of modifying and using the anti-PD-1 antibodies are described,e.g., in US20170247454, US 20170081409 A1, US 20170044259 A1, and US20160159905 A1; each of which is incorporated herein by reference in itsentirety.

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain atleast one (e.g., one, two, three, or four) of the antibodies orantigen-binding fragments described herein. Two or more (e.g., two,three, or four) of any of the antibodies or antigen-binding fragmentsdescribed herein can be present in a pharmaceutical composition in anycombination. The pharmaceutical compositions may be formulated in anymanner known in the art.

Pharmaceutical compositions are formulated to be compatible with theirintended route of administration (e.g., intravenous, intraarterial,intramuscular, intradermal, subcutaneous, or intraperitoneal). Thecompositions can include a sterile diluent (e.g., sterile water orsaline), a fixed oil, polyethylene glycol, glycerine, propylene glycolor other synthetic solvents, antibacterial or antifungal agents, such asbenzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like, antioxidants, such as ascorbic acid or sodiumbisulfite, chelating agents, such as ethylenediaminetetraacetic acid,buffers, such as acetates, citrates, or phosphates, and isotonic agents,such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol orsorbitol), or salts (e.g., sodium chloride), or any combination thereof.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers (see, e.g., U.S. Pat. No. 4,522,811). Preparations of thecompositions can be formulated and enclosed in ampules, disposablesyringes, or multiple dose vials. Where required (as in, for example,injectable formulations), proper fluidity can be maintained by, forexample, the use of a coating, such as lecithin, or a surfactant.Absorption of the antibody or antigen-binding fragment thereof can beprolonged by including an agent that delays absorption (e.g., aluminummonostearate and gelatin). Alternatively, controlled release can beachieved by implants and microencapsulated delivery systems, which caninclude biodegradable, biocompatible polymers (e.g., ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Compositions containing one or more of any of the antibodies orantigen-binding fragments described herein can be formulated forparenteral (e.g., intravenous, intraarterial, intramuscular,intradermal, subcutaneous, or intraperitoneal) administration in dosageunit form (i.e., physically discrete units containing a predeterminedquantity of active compound for ease of administration and uniformity ofdosage).

Toxicity and therapeutic efficacy of compositions can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals (e.g., monkeys). One can, for example, determine the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population): the therapeuticindex being the ratio of LD50:ED50. Agents that exhibit high therapeuticindices are preferred. Where an agent exhibits an undesirable sideeffect, care should be taken to minimize potential damage (i.e., reduceunwanted side effects). Toxicity and therapeutic efficacy can bedetermined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used informulating an appropriate dosage of any given agent for use in asubject (e.g., a dog, a cat, or a panda). A therapeutically effectiveamount of the one or more (e.g., one, two, three, or four) antibodies orantigen-binding fragments thereof (e.g., any of the antibodies orantibody fragments described herein) will be an amount that treats thedisease in a subject (e.g., kills cancer cells) in a subject (e.g., asubject identified as having cancer), or a subject identified as beingat risk of developing the disease (e.g., a subject who has previouslydeveloped cancer but now has been cured), decreases the severity,frequency, and/or duration of one or more symptoms of a disease in asubject (e.g., a dog, a cat, or a panda). The effectiveness and dosingof any of the antibodies or antigen-binding fragments described hereincan be determined by a health care professional or veterinaryprofessional using methods known in the art, as well as by theobservation of one or more symptoms of disease in a subject (e.g., adog, a cat, or a panda). Certain factors may influence the dosage andtiming required to effectively treat a subject (e.g., the severity ofthe disease or disorder, previous treatments, the general health and/orage of the subject, and the presence of other diseases).

Exemplary doses include milligram or microgram amounts of any of theantibodies or antigen-binding fragments described herein per kilogram ofthe subject's weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; or about 1μg/kg to about 50 μg/kg). While these doses cover a broad range, one ofordinary skill in the art will understand that therapeutic agents,including antibodies and antigen-binding fragments thereof, vary intheir potency, and effective amounts can be determined by methods knownin the art. Typically, relatively low doses are administered at first,and the attending health care professional or veterinary professional(in the case of therapeutic application) or a researcher (when stillworking at the development stage) can subsequently and graduallyincrease the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, andthe half-life of the antibody or antibody fragment in vivo.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. The disclosurealso provides methods of manufacturing the antibodies or antigen bindingfragments thereof for various uses as described herein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Generating Mouse Anti-dPD-1 Antibodies

To generate mouse antibodies against canine PD-1 (dPD-1; SEQ ID NO: 41),6-8 weeks old female BALB/c mice were immunized with canine PD-1.Anti-dPD-1 antibodies were collected by the methods as described belowand shown in FIG. 1A and FIG. 1B.

Immunization of Mice

6-8 weeks old female BALB/c mice were immunized with His-tagged caninePD-1 proteins at 20 μg/mouse at a concentration of 100 μg/ml. TheHis-tagged canine PD-1 proteins were emulsified with adjuvant andinjected at four positions on the back of the mice. For the firstsubcutaneous (s.c.) injection, the diluted antigen was emulsified withComplete Freund's Adjuvant (CFA) in equal volume. In the followingsubcutaneous injections, the protein was emulsified with IncompleteFreund's Adjuvant (IFA) in equal volume. Three days after the thirdinjection or the booster immunization, blood (serum) was collected andanalyzed for antibody titer using ELISA.

In another experiment, 6-8 weeks old female BALB/c mice were immunizedby injecting the expression plasmid encoding canine PD-1 into the mice.The plasmids encoding the antigen were injected into the tibialisanterior muscle (intramuscular injection; i.m. injection) of the mice byusing gene guns at the concentration of 1000 μg/ul at 60 μg per mouse.At least four injections were performed with at least 14 days betweentwo injections. Blood (serum) was collected seven days after the lastimmunization and the serum was tested for antibody titer by ELISA.

Procedures to enhance immunization were also performed at least fourteendays after the previous immunization (either by injecting the plasmid orby injecting the proteins). CHO cells that express PD-1 antigen on thesurface were intravenously injected into the mice through tail veins.Spleen was then collected four days after the injection.

Fusion of SP2/0 Cells and Spleen Cells

Spleen tissues were grinded. Spleen cells were first selected by CD3FMicrobeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0cells. The cells were then plated in 96-well plates withhypoxanthine-aminopterin-thymidine (HAT) medium.

Primary Screening of Hybridoma

Primary screening of the hybridoma supernatant in the 96-well plates wasperformed using Fluorescence-Activated Cell Sorting (FACS) pursuant tostandard procedures. Chinese hamster ovary (CHO) cells were added to96-well plates (2×10⁴ cells per well) before the screening. 50 μl ofsupernatant and human Fc-tagged canine PD-L1 proteins was used. Theantibodies that were used in experiments were

1) Fluorescein (PE)-conjugated AffiniPure F(ab)2 Fragment GoatAnti-Mouse IgG, Fc γ Fragment Specific, and

2) Fluorescein (FITC)-conjugated AffiniPure F(ab)2 Fragment GoatAnti-Human IgG, Fc γ Fragment Specific.

Sub-Cloning

Sub-cloning was performed using ClonePix2. In short, the positive wellsidentified during the primary screening were transferred to semisolidmedium, and IgG positive clones were identified and tested. FITCanti-mouse IgG Fc antibody was used.

Ascites Fluid Antibodies

1×10⁶ positive hybridoma cells were injected intraperitoneally to B-NDG®mice (Beijing Biocytogen, Beijing, China). Monoclonal antibodies wereproduced by growing hybridoma cells within the peritoneal cavity of themouse. The hybridoma cells multiplied and produced ascites fluid in theabdomens of the mice. The fluid contained a high concentration ofantibody which can be harvested for later use.

Purification of Antibodies

Antibodies in ascites fluid were purified using GE AKTA proteinchromatography (GE Healthcare, Chicago, Ill., United States). 13-1B9(“1B9”), 12-4A7 (“4A7”), and 12-1D8 (“1D8”) were among the mouseantibodies produced by the methods described above.

The VH, VL and CDR regions of the antibodies were determined. The heavychain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acidsequences of 1B9 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQID NOs: 19-24 (Chothia numbering).

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3amino acid sequences of 4A7 are shown in SEQ ID NOs: 7-12 (Kabatnumbering) or SEQ ID NOs: 25-30 (Chothia numbering).

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3amino acid sequences of 1D8 are shown in SEQ ID NOs: 13-18 (Kabatnumbering) or SEQ ID NOs: 31-36 (Chothia numbering).

Chimeric Antibodies

Based on the sequences of heavy chain and light chain variable regionsof 1B9, 4A7, and 1D8, chimeric anti-dPD-1 antibodies including1B9-mHvKv-dIgG4, 4A7-mHvKv-IgG4, and 1D8-mHvKv-IgG4 were generated.These chimeric antibodies have the heavy chain variable domain and thelight chain variable domain from the corresponding mouse anti-dPD-1antibodies, with the constant domains from canine IgG4 antibody(including, e.g., the CL, CH1, CH2, and CH3 domains). The sequences forconstant domains for canine IgG4 heavy chain is shown in SEQ ID NO: 69.The sequences for constant domain of canine IgG light chain is shown inSEQ ID NO: 70.

Example 2. Caninization of the Mice Antibodies

The starting point for caninization was the mouse antibodies (e.g., 1B9and 1D8). The amino acid sequences for the heavy chain variable regionand the light chain variable region of these mouse antibodies weredetermined.

Three caninized heavy chain variable region variants (SEQ ID NOs: 49-51)and three caninized light chain variable region variants (SEQ ID NOs:52-54) for 1B9 were constructed, containing different modifications orsubstitutions.

Three caninized heavy chain variable region variants (SEQ ID NOs: 42-44)and four caninized light chain variable region variants (SEQ ID NOs:45-48) for 1D8 were constructed, containing different modifications orsubstitutions.

These caninized heavy chain variable region variants can be combinedwith any of the light chain variable region variants based on the samemouse antibody. For example, 1B9-H1 (SEQ ID NO: 49) can be combined withany caninized light chain variable region variant based on the samemouse antibody 1B9 (e.g., 1B9-K3 (SEQ ID NO: 54)), and the antibody islabeled accordingly (e.g., 1B9-H1K3).

The caninized antibodies can have canine IgG antibody constant domains(including, e.g., the CL, CH1, CH2, and CH3 domains). For example,1B9-H1K1-IgG4 is based on the mouse antibody 1B9 and has the caninizedheavy chain variable domain H1 (SEQ ID NO: 49) and caninized light chainvariable domain K1 (SEQ ID NO: 52).

The name and the sequences of the chimeric anti-PD-1 antibodies and thecaninized anti-PD-1 antibodies are summarized in the table below.

TABLE 1 VH VL SEQ ID SEQ ID Type Antibody name NO: NO: Constant regionsChimeric 1B9-mHvKv-IgG4 59 60 Canine IgG4 antibody based on 1B9Caninized 1B9-H1K1-IgG4 49 52 Canine IgG4 antibodies 1B9-H1K2-IgG4 49 53Canine IgG4 based on 1B9 1B9-H1K3-IgG4 49 54 Canine IgG4 1B9-H2K1-IgG450 52 Canine IgG4 1B9-H2K2-IgG4 50 53 Canine IgG4 1B9-H2K3-IgG4 50 54Canine IgG4 1B9-H3K1-IgG4 51 52 Canine IgG4 1B9-H3K2-IgG4 51 53 CanineIgG4 1B9-H3K3-IgG4 51 54 Canine IgG4 Chimeric 4A7-mHvKv-IgG4 55 56Canine IgG4 antibody based on 4A7 Chimeric 1D8-mHvKv-IgG4 57 58 CanineIgG4 antibody based on 1D8 Caninized 1D8-H1K1-IgG4 42 45 Canine IgG4antibodies 1D8-H1K2-IgG4 42 46 Canine IgG4 based on 1D8 1D8-H1K3-IgG4 4247 Canine IgG4 1D8-H1K4-IgG4 42 48 Canine IgG4 1D8-H2K1-IgG4 43 45Canine IgG4 1D8-H2K2-IgG4 43 46 Canine IgG4 1D8-H2K3-IgG4 43 47 CanineIgG4 1D8-H2K4-IgG4 43 48 Canine IgG4 1D8-H3K1-IgG4 44 45 Canine IgG41D8-H3K2-IgG4 44 46 Canine IgG4 1D8-H3K3-IgG4 44 47 Canine IgG41D8-H3K4-IgG4 44 48 Canine IgG4

Example 3. In Vitro Testing of the Mouse Anti-dPD-1 Antibodies: Blockingthe Binding of Canine PD-1 (dPD-1) and Canine PD-L1 (dPD-L1)

Blocking assays were performed to determine whether the anti-dPD-1antibodies can block the binding between dPD-1 and its ligand dPD-L1.

200 μL of canine PD-L1 Protein (Fc Tag) (250 μg/mL) (Sino Biological,Beijing; Cat #70110-1D02H) was mixed with 1.94 μL of EZ-Link®Sulfo-NHS-LC-Biotin (10 mM). The mixture was then desalted by PDMiniTrap G-10 column and was stored at −20° C. for future use.

The anti-dPD-1 chimeric antibodies were collected from mouse ascitesfluid and purified by chromatography. 25 μl CHO cells transientlytransfected with canine PD-1 were added to each well in a plate. Thepurified antibodies were titrated to the desired final concentrations,and were added to each well at 25 μl per well at 4° C. and incubated for30 minutes.

Bitoin labeled dPD-L1 was added to each well (with a final concentrationof 5 μg/ml in each well). The cells, Bitoin labeled dPD-L1 and theantibodies were incubated at 4° C. for 30 minutes.

After being washed with phosphate-buffered saline (PBS) twice (1200 rpm,5 minutes), 50 μl of PE-labeled Streptavidin (Streptavidin-PE) at 1:100dilution was added into each well, and incubated for 30 minutes at 4°C., followed by PBS wash (1200 rpm, 5 minutes). 20 μl of PBS was added.The signals for PE was detected by flow cytometry (Intellicyt).

As shown in FIG. 3, when the concentration of the chimeric anti-dPD-1antibodies 4A7-mHvKv-dlgG4, 1D8-mHvKv-dlgG4, and 1B9-mHvKv-dlgG4decreased, the signal for PE increased (x axis), suggesting that thebinding between canine PD-1 and canine PD-L1 was blocked by the threeanti-dPD-1 antibodies.

Example 4. Cross-Reactivity of Anti-dPD-1 Antibodies Against Human,Mouse, and Dog-Mouse Chimeric PD-1, Panda PD-1 and Feline PD-1

In each experiment, the CHO cells were transfected with human PD-1(hPD-1, SEQ ID NO: 37), mouse PD-1 (mPD-1, SEQ ID NO: 38), chimeric(mouse and dog) PD-1 (chidPD-1, SEQ ID NO: 40), or panda PD-1 (pPD-1,SEQ ID NO: 61)

25 μl CHO cells were added to each well. 25 μl purified anti-dPD-1chimeric antibodies (10 μg/ml) (4A7-mHvKv-dlgG4, 1D8-mHvKv-dlgG4, or1B9-mHvKv-dlgG4) were added to each well and were incubated at 4° C. for30 minutes.

After being washed with PBS (1200 rmp, 5 min) twice, FITC labeledanti-dog IgG Fc antibody (anti-dIgG Fc-FITC) was added into each well1:100 dilution, followed by incubating at 4° C. for 30 minutes, and thenPBS wash (1200 rmp, 5 min). The signals for FITC were determined by flowcytometry.

As shown in FIG. 4, 4A7-mHvKv-dlgG4, 1D8-mHvKv-dlgG4, and1B9-mHvKv-dlgG4 did not cross react with human PD-1 or mouse PD-1, buthad strong binding activity with chidPD-1. With respect to panda PD-1,4A7-mHvKv-dlgG4 and 1D8-mHvKv-dlgG4 had some cross-reactivity withpPD-1, and 1B9-mHvKv-dlgG4 had strong binding activity with pPD-1 (FIG.5). In FIG. 4 and FIG. 5, NC stands for negative control.

The CHO cells were transfected with an expression vector that expressesfeline PD-1 (cPD-1, SEQ ID NO: 62) and EGFP. 25 μl purified anti-dPD-1chimeric antibodies (10 μg/ml) were added to each well and wereincubated at 4° C. for 30 minutes. After being washed with PBS (1200rmp, 5 min) twice, Alexa Fluor 647 labeled anti-dog IgG Fc antibody(anti-dIgG Fc-647) was added into each well 1:500 dilution, followed byincubating at 4° C. for 30 minutes, and then PBS wash (1200 rmp, 5 min).The signals for GFP and FITC were determined by flow cytometry. Theresults show that 1B9-mHvKv-dlgG4 did not bind to feline PD-1. However,4A7-mHvKv-dlgG4 and 1D8-mHvKv-dlgG4 had some binding activity withfeline PD-1 (FIG. 6).

Example 5. EC50 for Binding Activity Against Panda PD-1, Canine PD-1,and Feline PD-1

In each experiment, the CHO cells were transfected with panda PD-1,canine PD-1, or feline PD-1.

25 μl CHO cells were added to each well. 25 μl purified anti-dPD-1chimeric antibodies with different concentrations were added to eachwell and were incubated at 4° C. for 30 minutes.

After being washed with PBS (1200 rmp, 5 min) twice, 50 μl of AlexaFluor 647 labeled anti-dog IgG Fc antibody (anti-dIgG Fc-647) was addedinto each well at 1:1000 dilution, followed by incubating at 4° C. for30 minutes, and then PBS wash (1200 rmp, 5 min). 30 μl of PBS was thenadded. The signals for Alexa Fluor 647 were determined by flowcytometry. And the curve for the mean of fluorescence intensity (MFI)was used to determine EC50.

In FIG. 7, the CHO cells were transfected with panda PD-1. FIG. 7 showsthe MFI at different concentrations of 4A7-mHvKv-dlgG4 and1B9-mHvKv-dlgG4. EC50 were determined. The result shows that1B9-mHvKv-dlgG4 has strong binding activity with panda PD-1.

TABLE 2 Antibody EC50 (nM) for panda PD-1 4A7-mHvKv-dIgG4 385.51B9-mHvKv-dIgG4 2.866

In FIG. 8, the CHO cells were transfected with canine PD-1. FIG. 8 showsthe MFI at different concentrations of 4A7-mHvKv-dlgG4 and1B9-mHvKv-dlgG4. EC50 were determined. The result shows that bothantibodies have strong binding activity with canine PD-1.

TABLE 3 Antibody EC50 (nM) for canine PD-1 4A7-mHvKv-dIgG4 4.811B9-mHvKv-dIgG4 4.52

In FIG. 9, the CHO cells were transfected with an expression vector thatexpresses feline PD-1 and EGFP. 25 μl purified anti-dPD-1 chimericantibodies with different concentrations were added to each well. Afterbeing washed with PBS (1200 rmp, 5 min) twice, 50 μl of Alexa Fluor 647labeled anti-dog IgG Fc antibody (anti-dIgG Fc-647) was added into eachwell at 1:500 dilution, followed by incubating at 4° C. for 30 minutes,and then PBS wash (1200 rmp, 5 min). 30 μl of PBS was then added. FIG. 9shows the MFI at different concentrations of 4A7-mHvKv-dlgG4 and1D8-mHvKv-dlgG4. EC50 were also determined from the data. The resultsshow that both antibodies have strong binding activity with feline PD-1,and the EC50 for 4A7-mHvKv-dIgG4 is better than 1D8-mHvKv-dlgG4.

TABLE 4 Antibody EC50 (nM) for feline PD-1 4A7-mHvKv-dlgG4 1.528 1D8-mHvKv-dlgG4 7.291

Example 6. Binding Affinity of Anti-dPD-1 Antibodies

The binding affinity of the anti-dPD-1 antibodies were measured usingsurface plasmon resonance (SPR) using Biacore (Biacore, INC, PiscatawayN.J.)

Histidine-tagged canine PD-1 proteins (dPD-1-His) were diluted to thefinal concentration of 3.34 μg/ml and fixed to the CM5 chip with AmineCoupling Kit (GE Healthcare; Cat #BR100050) to achieve to a desiredprotein density (about 90 response units (RU)).

Anti-dPD-1 antibodies at the concentration of 40, 20, 10, 5, 2.5, 1.25,or 0.625 nM were injected at 30 μL/min for 120-240 seconds. Dissociationwas monitored for 600 seconds. The chip was regenerated after the lastinjection of each titration with Glycine (pH 1.5, 30 μL/min for 16seconds). The result for 1D8-mHvKv-dlgG4 is shown in FIG. 10 as anexample.

Kinetic association rates (kon) and dissociation rates (koff) wereobtained simultaneously by fitting the data globally to a 1:1 Langmuirbinding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994.Methods Enzymology 6. 99-110) using Biacore T200 Evaluation Software3.0. Affinities were deduced from the quotient of the kinetic rateconstants (KD=koff/kon).

As a person of ordinary skill in the art would understand, the samemethod with appropriate adjustments for parameters (e.g., antibodyconcentration) was performed for each tested antibody. The results forthe tested antibodies are summarized in the table below.

TABLE 5 Association Dissociation Anti-dPD-1 rate kon rate koff Affinityantibodies (1/Ms) (1/s) KD (M) 1D8-H1K1-dIgG4 6.75E+05 3.09E−03 4.57E−091D8-H1K2-dIgG4 5.50E+05 2.48E−03 4.50E−09 1D8-H2K1-dIgG4 6.50E+053.48E−03 5.36E−09 1D8-H2K2-dIgG4 6.04E+05 2.62E−03 4.34E−091D8-mHVKV-dIgG4 1.44E+06 1.35E−04 9.40E−11 1B9-H1K1-IgG4 4.24E+054.54E−04 1.07E−09 1B9-H1K2-IgG4 4.75E+05 4.90E−04 1.03E−09 1B9-H2K1-IgG45.91E+05 4.54E−04 7.68E−10 1B9-H2K2-IgG4 5.77E+05 4.30E−04 7.45E−101B9-mHVKV-dIgG4 1.010E+06 1.260E−04 1.250E−10 4A7-mHVKV-dIgG4 5.25E+051.52E−04 2.90E−10

Example 7. In Vivo Testing of Chimeric and Caninized Anti-dPD-1Antibodies

In order to test the anti-dPD-1 antibodies in vivo and to predict theeffects of these antibodies in dogs, a caninized PD-1 mouse model wasgenerated. The caninized PD-1 mouse model was engineered to express achimeric PD-1 protein (SEQ ID NO: 40) wherein a part of theextracellular region of the mouse PD-1 protein was replaced with thecorresponding canine PD-1 extracellular region. The caninized mousemodel provides a new tool for testing new therapeutic treatments in aclinical setting by significantly decreasing the difference betweenclinical outcome in dogs and in ordinary mice expressing mouse PD-1.

The anti-dPD-1 antibodies were tested for their effect on tumor growthin vivo in a model of colon carcinoma. MC-38 cancer tumor cells (colonadenocarcinoma cell) were injected subcutaneously in the mice. When thetumors in the mice reached a volume of 150±50 mm³, the mice wererandomly placed into different groups based on the volume of the tumor(five mice in each group).

The mice were then injected with physiological saline (PS) andanti-dPD-1 antibodies by intraperitoneal administration as indicated inthe table below. The antibody was given on the second day and the fifthday of each week. Most groups had 6 administrations in total. G12 onlyhad 2 administrations in total.

TABLE 6 Dosage Total No. of Group No. of mice Antibodies (mg/kg) RouteFrequency administration G1 4 PS (control) — i.p. Day 2, 5/wk 6 G2 41B9-H1K1-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G3 4 1B9-H1K2-dIgG4 3 mg/kgi.p. Day 2, 5/wk 6 G4 4 1B9-H2K1-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G5 41B9-H2K2-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G6 4 1D8-H1K1-dIgG4 3 mg/kgi.p. Day 2, 5/wk 6 G7 4 1D8-H1K2-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G8 41D8-H2K1-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G9 4 1D8-H2K2-dIgG4 3 mg/kgi.p. Day 2, 5/wk 6 G10 4 1D8-mHvKv-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G114 1B9-m HvKv-dIgG4 3 mg/kg i.p. Day 2, 5/wk 6 G12 4 4A7-mHvKv-dIgG4 3mg/kg i.p. Day 2, 5/wk 2

The injected volume was calculated based on the weight of the mouse at 3mg/kg. The length of the long axis and the short axis of the tumor weremeasured and the volume of the tumor was calculated as 0.5×(longaxis)×(short axis)². The weight of the mice was also measured before theinjection, when the mice were placed into different groups (before thefirst antibody injection), twice a week during the treatment period, andbefore euthanization.

The tumor growth inhibition percentage (TGI %) was calculated using thefollowing formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100. Ti is the averagetumor volume in the treatment group on day i. T0 is the average tumorvolume in the treatment group on day zero. Vi is the average tumorvolume in the control group on day i. V0 is the average tumor volume inthe control group on day zero.

T-test was performed for statistical analysis. A TGI % higher than 60%indicates strong suppression of tumor growth. P<0.05 is a threshold toindicate significant difference.

The weight of the mice was monitored during the entire treatment period.The weight of mice in different groups all increased (FIG. 11, and FIG.12). No obvious difference in weight was observed among the groups. Theresults showed that the tested antibodies were well tolerated and werenot toxic to the mice.

The tumor size in most groups treated with anti-dPD-1 antibodiesincreased to a lesser extent compared to the control group (FIGS.13-15). The TGI % at day 24 (24 days after grouping) was also calculatedas shown in the table below.

TABLE 7 Tumor volume(mm3) P value Day Day Day Day Body Tumor 0 10 17 24Survival TGI % weight Volume Control G1 137 ± 903 ± 1605 ±  2956 ±  4/4n.a. n.a. n.a. 16 225  537 932 Treat G2 142 ± 315 ± 527 ± 952 ± 4/471.3% 0.170 0.082 16 79  98 230 G3 121 ± 185 ± 354 ± 558 ± 4/4 84.5%0.042 0.044  9 27  69 138 G4 130 ± 258 ± 326 ± 735 ± 4/4 78.6% 0.0050.063 20 80 100 284 G5 119 ± 296 ± 304 ± 940 ± 4/4 70.9% 0.042 0.116 1467 110 583 G6 131 ± 796 ± 1085 ±  2687 ±  4/4  9.3% 0.452 0.822 22 111 207 663 G7 146 ± 350 ± 667 ± 1431 ±  4/4 54.4% 0.088 0.297  9 172  415954 G8 134 ± 278 ± 371 ± 723 ± 4/4 79.1% 0.004 0.060 21 62 149 255 G9140 ± 278 ± 405 ± 970 ± 4/4 70.5% 0.118 0.102 16 57 138 440 G10 124 ±213 ± 290 ± 505 ± 4/4 86.5% 0.002 0.041 18 37  63 146 G11 134 ± 284 ±383 ± 766 ± 4/4 77.6% 0.027 0.064 20 68  93 253 G12 141 ± 184 ± 314 ±990 ± 4/4 69.9% 0.012 0.096 14  6  69 358

The results above show that all anti-dPD-1 antibodies can inhibit tumorgrowth to some extent. Among them, G3 (1B9-H1K2-dIgG4) and G10(1D8-mHvKv-dIgG4) had the highest tumor growth inhibition percentage(TGI %). In addition, animals in G12 only received 2 administrations of4A7-mHvKv-dIgG4, and tumor inhibitory effects were still remarkable.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An antibody or antigen-binding fragment thereof that binds to PD-1(Programmed Cell Death Protein 1) comprising: a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,and 3, wherein the VH CDR1 region comprises an amino acid sequence thatis at least 80% identical to a selected VH CDR1 amino acid sequence, theVH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3region comprises an amino acid sequence that is at least 80% identicalto a selected VH CDR3 amino acid sequence; and a light chain variableregion (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 regioncomprises an amino acid sequence that is at least 80% identical to aselected VL CDR1 amino acid sequence, the VL CDR2 region comprises anamino acid sequence that is at least 80% identical to a selected VL CDR2amino acid sequence, and the VL CDR3 region comprises an amino acidsequence that is at least 80% identical to a selected VL CDR3 amino acidsequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequencesand the selected VL CDRs, 1, 2, and 3 amino acid sequences are one ofthe following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences areset forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6,respectively; (2) the selected VH CDRs 1, 2, 3 amino acid sequences areset forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12,respectively; and (3) the selected VH CDRs 1, 2, 3 amino acid sequencesare set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selectedVL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16,17, 18, respectively.
 2. The antibody or antigen-binding fragmentthereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the aminoacid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, andthe VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth inSEQ ID NOs: 4, 5, and 6, respectively.
 3. The antibody orantigen-binding fragment thereof of claim 1, wherein the VH comprisesCDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7,8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the aminoacid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. 4.The antibody or antigen-binding fragment thereof of claim 1, wherein theVH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQID NOs: 13, 14, 15, respectively, and the VL comprises CDRs 1, 2, 3 withthe amino acid sequences set forth in SEQ ID NOs: 16, 17, 18,respectively.
 5. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment specificallybinds to canine PD-1.
 6. The antibody or antigen-binding fragmentthereof of claim 1, wherein the antibody or antigen-binding fragmentbinds to PD-1 of Ailuropoda melanoleuca (giant panda) or Felis catus(domestic cat).
 7. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment is a caninizedantibody or antigen-binding fragment thereof.
 8. A nucleic acidcomprising a polynucleotide encoding a polypeptide comprising: (1) animmunoglobulin heavy chain or a fragment thereof comprising a heavychain variable region (VH) comprising complementarity determiningregions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forthin SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, whenpaired with a light chain variable region (VL) comprising the amino acidsequence set forth in SEQ ID NO: 52, 53, 54, or 60, binds to PD-1; (2)an immunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, whenpaired with a VH comprising the amino acid sequence set forth in SEQ IDNO: 49, 50, 51, or 59, binds to PD-1; (3) an immunoglobulin heavy chainor a fragment thereof comprising a heavy chain variable region (VH)comprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, whenpaired with a light chain variable region (VL) comprising the amino acidsequence set forth in SEQ ID NO: 56, binds to PD-1; (4) animmunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL,when paired with a VH comprising the amino acid sequence set forth inSEQ ID NO: 55, binds to PD-1; (5) an immunoglobulin heavy chain or afragment thereof comprising a heavy chain variable region (VH)comprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 13, 14, 15, respectively, and wherein the VH, whenpaired with a light chain variable region (VL) comprising the amino acidsequence set forth in SEQ ID NO: 45, 46, 47, 48 or 58 binds to PD-1; or(6) an immunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 16, 17, 18, respectively, and wherein the VL, whenpaired with a VH comprising the amino acid sequence set forth in SEQ IDNO: 42, 43, 44 or 57 binds to PD-1. 9.-18. (canceled)
 19. A vectorcomprising one or more of the nucleic acids of claim
 7. 20.-21.(canceled)
 22. A cell comprising one or more of the nucleic acids ofclaim
 7. 23.-26. (canceled)
 27. A method of producing an antibody or anantigen-binding fragment thereof, the method comprising culturing thecell of claim 22 under conditions sufficient for the cell to produce theantibody or the antigen-binding fragment; and.
 28. An antibody orantigen-binding fragment thereof that binds to PD-1 comprising a heavychain variable region (VH) comprising an amino acid sequence that is atleast 90% identical to a selected VH sequence, and a light chainvariable region (VL) comprising an amino acid sequence that is at least90% identical to a selected VL sequence, wherein the selected VHsequence and the selected VL sequence are one of the following: (1) theselected VH sequence is SEQ ID NOs: 49, 50, 51, or 59, and the selectedVL sequence is SEQ ID NOs: 52, 53, 54, or 60; (2) the selected VHsequence is SEQ ID NOs: 42, 43, 44, or 57, and the selected VL sequenceis SEQ ID NOs: 45, 46, 47, 48, or 58; and (3) the selected VH sequenceis SEQ ID NO: 55, and the selected VL sequence is SEQ ID NO:
 56. 29. Theantibody or antigen-binding fragment thereof of claim 28, wherein (1)the VH comprises the sequence of SEQ ID NO: 49 and the VL comprises thesequence of SEQ ID NO: 53: (2) the VH comprises the sequence of SEQ IDNO: 43 and the VL comprises the sequence of SEQ ID NO: 45: (3) the VHcomprises the sequence of SEQ ID NO: 43 and the VL comprises thesequence of SEQ ID NO: 46: (4) the VH comprises the sequence of SEQ IDNO: 49 and the VL comprises the sequence of SEQ ID NO: 52: (5) the VHcomprises the sequence of SEQ ID NO: 50 and the VL comprises thesequence of SEQ ID NO: 52: or (6) the VH comprises the sequence of SEQID NO: 50 and the VL comprises the sequence of SEQ ID NO:
 53. 30.-33.(canceled)
 34. An antibody-drug conjugate comprising the antibody orantigen-binding fragment thereof of claim 1 covalently bound to atherapeutic agent.
 35. (canceled)
 36. A method of treating a subjecthaving cancer, the method comprising administering a therapeuticallyeffective amount of a composition comprising the antibody orantigen-binding fragment thereof of claim 1, to the subject.
 37. Themethod of claim 36, wherein the subject has a solid tumor, unresectablemelanoma, metastatic melanoma, non-small cell lung cancer (NSCLC),squamous cell carcinoma of the head and neck (SCCHN), head and neckcancer, renal cell carcinoma (RCC), melanoma, bladder cancer, gastriccancer, urothelial cancer, Merkel-cell carcinoma, triple-negative breastcancer (TNBC), or colorectal carcinoma. 38.-39. (canceled)
 40. A methodof decreasing the rate of tumor growth, the method comprising contactinga tumor cell with an effective amount of a composition comprising anantibody or antigen-binding fragment thereof of claim
 1. 41. A method ofkilling a tumor cell, the method comprising contacting a tumor cell withan effective amount of a composition comprising the antibody orantigen-binding fragment thereof of claim
 1. 42. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereofof claim 1, and a pharmaceutically acceptable carrier.
 43. (canceled)44. An antibody or antigen-binding fragment thereof that binds to PD-1,comprising a heavy chain variable region comprising VH CDRs 1, 2, 3, anda light chain variable region comprising VL CDRs 1, 2, 3, wherein the VHCDRs 1, 2, 3 and the VL CDRs 1, 2, 3 are identical to VH CDRs 1, 2, 3and VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereofof claim 28.